AU726058B2 - Pyrimidine carboxylates and related compounds and methods for treating inflammatory conditions - Google Patents

Description

WO 97/09325 PCT/US96/14089 1 Description PYRIMIDINE CARBOXYLATES AND RELATED COMPOUNDS AND METHODS FOR TREATING INFLAMMATORY CONDITIONS Technical Field The present invention relates generally to compounds that block intracellular signal transduction and activation of transcription factors, and to methods for preventing or treating immunoinflammatory and autoimmune diseases.

Background of the Invention Signals necessary for cell growth, differentiation, response to bioregulatory molecules, infectious agents and physiological stress involve changes in the rates of gene expression. The ability to respond appropriately to such signaling events challenge the survival of the cell and ultimately the organism. Perturbations in the normal regulation of these specific genetic responses can result in pathogenic events which lead to acute and chronic disease.

In certain autoimmune diseases or chronic inflammatory states, continuous activation of T-cells eventually leads to a self-perpetuating destruction of normal tissues or organs. This is caused by the induction of adhesion molecules, chemotaxis of leukocytes, activation of leukocytes and the production of mediators of inflammation. All of these events are regulated at the level of transcription for the production of new proteins, including cytokines. The production of cytokines, as well as a number of other cellular regulators, is controlled by a family of proteins known as transcription factors (TFs). These transcription factors, when activated, bind to specific regions on the DNA and act as molecular switches or messengers to induce or upregulate gene expression. The activation of these TFs is caused by a variety of external signals including physiological stress, infectious agents and other bioregulatory molecules. Once the plasma membrane receptors are activated, a cascade of protein kinases and second messengers are induced which, in turn, result in the production of WO 97/09325 PCT/US96/14089 2 RNA transcripts. The end result is the production of proinflammatory proteins via translation and processing of the RNA transcripts.

This activation system can, at times, be very robust. For example, a specific set of external signals could result in a single transcription factor to induce many proteins responsible for a given disease. Therefore, regulating this process by disrupting the production of activated TF(s) has the potential to attenuate the production of the associated pathological proteins, thereby halting or reversing the course of the disease.

Two transcription factors, NFKB and AP-1, have been shown to regulate the production of many proinflammatory cytokines and related proteins that are elevated in immunoinflammatory diseases. These TFs regulate interleukin-1 (IL-1), interleukin-2 tumor necrosis factor-ca (TNFa), interleukin-6 (IL-6) and interleukin-8 (IL-8) levels in a variety of cell types. For example, NFKB and other related complexes are involved in the rapid induction of genes whose products function in protective and proliferative responses upon exposure of cells to external stimuli.

Similarly, AP- has a significant role in the regulation of interleukin-2 (IL-2) and tumor necrosis factor-a (TNF-a) transcription during T-cell activation. In addition, TNF-a and IL-1 are strong activators of collagenase, gelatinase and stromelysin gene expression, which require a single AP-1 binding site in the promoter region of these genes. Therefore, an inhibitor of NFKB and/or AP-1 activation would coordinately repress the activities of a series of proteinases. In addition, cell adhesion molecules are also controlled by these TFs. All of these proteins have been shown to play a role in diseases, including osteoarthritis, transplant rejection, ischemia, reperfusion injury, trauma, certain cancers and viral disorders, and autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, glomerulonephritis, lupus and juvenile diabetes. In summary, the role of these TFs is to act as a transducer for certain stimuli that lead to immune, inflammatory, and acute phase responses.

Since many diseases are caused by the inappropriate production of proteins, conventional therapeutic approaches have focused on inhibiting function or activity of individual effector proteins. These treatments have not always proved to be effective and, at times, are associated with many undesirable side effects. Therefore, there is a need for new therapies for the prevention and/or treatment of immunoinflammatory and autoimmune diseases. More specifically, there is a need for compounds that prevent, preferably by inhibiting transcription at an early stage, the production of proteins associated with immunoinflammatory and autoimmune diseases.

Furthermore, these compounds should inhibit the kinase(s) that regulate the activation of TFs such as NFcB and AP-1. The present invention fulfills these needs and provides further related advantages.

Summary of the Invention In brief, this invention is directed to compounds that block the activation of transcription factors (TFs), particularly NFxB and AP-1, and are believed to function through inhibition of a family of specific kinases. This results in a decrease in a number 15 of proinflammatory proteins, including IL-1, IL-2, IL-8 and/or TNFa, which are responsible for tissue and organ damage associated with diseases such as rheumatoid arthritis, osteoarthritis, related autoimmune disorders and tissue rejection. Accordingly, 0' compounds of the present invention are useful in, for example, the prevention of organ and tissue rejection associated with transplantation. Furthermore, the compounds of 20 this invention also have utility in the prevention and/or treatment of immunoinflammatory and autoimmune diseases, as well as having general activity as anti-inflammatory agents.

In one aspect of this invention, there are provided compounds having the following general structure

R

R4 R6 N N

R

2 P:\OPER\PDB\70130-96 spci.doc-29/08/(0I -4including pharmaceutically and prophylactically acceptable salts thereof, wherein

R

2 is R 2 a when R 4 is R 4 a, and R 2 is R2b when R 4 is R4b; R2b and R4a, are selected from hydrogen, halogen and an unsubstituted or substituted C1.-alkyl, C 6 -12aryl, C 7 .12aralkyl, C 3 -1 2 heterocycle or C4.16heterocyclealkyl; R2a and R4b are selected from the following chemical moieties:

R

g 0 Rg 0 \N Rio N R1 H H R11 Rio S 10 R 5 is selected from -CH 2 0{C(=O)O} 0 o.R 7

-C(=O)OR

7 and

R

6 is selected from hydrogen, -CH 3

-CH

2

C

6

H

5 -F and -CF 3

R

7 is selected from hydrogen and an unsubstituted or substituted C 1 8 alkyl, C6-12aryl and C7-12aralkyl;

R

8 is an unsubstituted or substituted Ci.galkyl, C 6 -1 2 aryl or C7-1 2 aralkyl; R is selected from hydrogen 7 and an unsubstituted C 1 8 alkyl or C7-14aralkyl, wherein D is a direct bond, or -NH-; RIo and R 11 are the same or different and independently selected from hydrogen and an unsubstituted or substituted Cl.salkyl or C6-12aryl; and n is an integer from 0 to 4 and each occurrence of A is a substituent independently selected from halogen, -OH, -OR, -COOH, -COOR, -COR, -CONH 2

-NH

2 -NHR, -NRR, NO 2 -SH, -SR, -SOOR, -S03R and -SOR, where each occurrence of R is independently selected from an unsubstituted or substituted Ci.8alkyl, C6- 12 aryl or C 7 1 2 aralkyl.

In another aspect, there is provided a pharmaceutical composition containing one or more compounds of this invention in combination with a pharmaceutically or prophylactically acceptable carrier or diluent.

iRA1 In a further aspect, there are provided methods for preventing and/or reating inflammatory conditions by administering to a warm-blooded animal in need P:\OPER\PDB\70130-96 peci.doc-29//AX -4Athereof an effective amount of a compound of this invention. Such inflammatory conditions include both immunoinflammatory conditions and autoimmune diseases. In the practice of the disclosed methods, the compounds are preferably administered to the warmblooded animal in the form or a pharmaceutical composition.

The invention also provides for the use of compounds of the invention in the manufacture of a medicament for preventing and/or treating inflammatory conditions.

Brief Description of the Drawings Figures 1 and 2 illustrate reaction schemes for the synthesis of representative compounds of this invention.

Figure 3 illustrates the ability of a representative compound of this invention to inhibit the activation of NFKB and AP- 1.

*ii* Figure 4 illustrates the ability of a representative compound of this invention to inhibit IL-2 and IL-8.

o 15 Detailed Description of the Invention As mentioned above, the compounds of this invention block activation of transcription factors (TFs), and thus have utility as anti-inflammatory agents in general, and in the prevention and/or treatment of a variety of conditions, including (but not limited to) immunoinflammatory and autoimmune diseases. The compounds are believed to 20 function by inhibiting, at an early stage, transcription of deleterious proteins associated :i with such conditions or diseases. It is believed that this is achieved by inhibiting the kinase(s) that regulate the activation of TFs, such as NFKB and/or AP-1. By disrupting the production of these activated TFs, synthesis of pathological proteins, including proinflammatory cytokines, associated with a series of immunoinflammatory WO 97/09325 PCT/US96/14089 and autoimmune diseases are effectively blocked at a transcriptional level.

Accordingly, the compounds of this invention have activity in both the prevention and treatment of immunoinflammatory diseases such as rheumatoid arthritis, osteoarthritis and transplant rejection (tissue and organ), as well as autoimmune diseases such as multiple sclerosis.

The compounds of this invention are generally represented by the following structure R4

R

NN

R2

(I)

wherein

R

2

R

4 R, and R 6 are as defined below.

In structure above,

R

5 is selected from the following chemical moieties (ii) and (iii): 0,1 i R7 0 R r0 0' (ih) wherein

R

7 is selected from hydrogen and an unsubstituted or substituted C,-.alkyl,

C

6 12 aryl or C,.,aralkyl; and R, is an unsubstituted or substituted C,.,alkyl,

C

6 ,,,aryl or C, ~aralkyl. In a preferred embodiment, R, is a C,, 1 alkyl, and in a more preferred embodiment is selected from methyl and ethyl. Further, in a preferred embodiment, R, is selected from methyl and phenyl.

WO 97/09325 PCT/US96/14089 6 The compounds of this invention further include pharmaceutically and prophylactically acceptable salts of compounds of structure Compounds of structure may contain proton donating groups a carboxylic acid group) and/or proton accepting groups a group with a nitrogen atom having a free lone pair of electrons, such as an amine group), and the salts of compounds of structure may be formed and utilized in the practice of the invention. Thus, compounds of the invention may be in the form of a base addition salt a salt of a proton donating group) or in the form of an acid addition salt a salt of a proton accepting group), as well as the free acid or free base forms thereof.

Acid addition salts of a free base amino compound of the invention may be prepared by methods well known in the art, and may be formed from organic and inorganic acids. Suitable organic acids include acetic, ascorbic, benzenesulfonic, benzoic, fumaric, maleic, methanesulfonic, and succinic acids. Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, phosphoric and nitric acids. Base addition salts of a free acid carboxylic acid compound of the invention may also be prepared by methods well known in the art, and may be formed from organic and inorganic bases.

Thus, the compounds of this invention also include those salts derived from inorganic bases such as the hydroxide or other salt of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like, and organic bases such as substituted ammonium salts.

As used herein, the above terms have the following meaning: A "C,.,alkyl" is a straight chain or branched, cyclic or non-cyclic, saturated or unsaturated carbon chain containing from 1 to 8 carbon atoms. In one embodiment, the C,.,alkyl is a fully saturated, straight chain alkyl selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl and n-hexyl. In another embodiment, the C,_alkyl is a fully saturated cyclic alkyl selected from (but not limited to) cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylenecyclopropyl and methylenecyclohexyl. In still a further embodiment, the C, _alkyl is a fully saturated, branched alkyl selected from (but not limited to) isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl and isohexyl. In yet a WO 97/09325 PCT/US96/14089 7 further embodiment, the C,_,alkyl is an unsaturated straight chain alkyl selected from (but not limited to) ethylenyl, propylenyl, 1-butenyl, 1-pentenyl and 1-hexenyl.

A "C6 1 2 aryl" is an aromatic moiety containing from 6 to 12 carbon atoms. In one embodiment, the C 6 2 aryl is selected from (but not limited to) phenyl, tetralinyl, and napthalenyl. In a preferred embodiment, the C 6 12 aryl is phenyl.

A "C 7 2 aralkyl" is an arene containing from 7 to 12 carbon atoms, and has both aliphatic and aromatic units. In one embodiment, the C 7 2 ,aralkyl is selected from (but not limited to) benzyl, ethylbenzyl, propylbenzyl and isobutylbenzyl.

A "substituted" C,,alkyl,

C

6 12 aryl or C7 2 ,aralkyl is a C.

8 ,alkyl, C6 12 ary or C 7 2 aralkyl having one or more hydrogens replaced with a substituent selected from halogen (including -Cl, -Br and -OH, -OR, -COOH, -COOR,

-COR,

-CONH

2

-NH

2 -NHR, -NRR, -NO2, -SH, -SR, -SOOR,

-SO

3 R and -SOR, where each occurrence of R is independently selected from an unsubstituted or substituted C,_,alkyl,

C

6 12 aryl or 2 aralkyl as defined above. In one embodiment, the substituted C,,alkyl is a C,.ghaloalkyl including (but not limited to) -CF, and -C 2

F,.

In one embodiment of structure above, R, is R 2 a and R 4 is R 4 a. In this embodiment, R4a is selected from hydrogen, halogen and an unsubstituted or substituted C,.salkyl, C,,,aryl,

C,.

12 aralkyl, C3, 2 heterocycle or C 4 ,,,heterocyclealkyl; and R 2 a is selected from the following chemical moieties (iv) through (vii): R O

A

S I I Y R9

N

ONO WA iRo R9 0 RR9 0 0 H H Rio (iv) (vi) (vii) wherein R, is selected from hydrogen, and an unsubstituted

C,

8 alkyl or

C

7 .,,aralkyl; and Rio and are the same or different and independently selected from hydrogen and an unsubstituted or substituted C,,alkyl or C.

1 2 aryl; n is an integer from 0 to 4 and represents the number of substituents on the benzene ring of chemical moiety D represents a direct bond, or and each occurrence of A is independently WO 97/09325 PCT/US96/1 4089 8 selected from a substituent as identified above. In a preferred embodiment, D is a direct bond; R 9 is selected from hydrogen,

-CH

3

-CH

2

CH

3 and -CH 2

C

6

H

5 Rio and ar6 the same or different and independently selected from hydrogen,

-CH

3

-CF

3

-(CH

2 )1-5CH 3

-C

6

H

5

-CH

2

C

6 and a substituted phenyl or benzyl moiety; and n is 0.

In another embodiment of structure above, R 2 is R 2 b and R 4 is R 4 b. In this embodiment,

R

2 b is selected from hydrogen, halogen and an unsubstituted or substituted

C,.

8 alkyl, C6- 1 2 aryl, C7- 1 2 aralkyl,

C

3 1 2 heterocycle or C 4 -1 6 heterocyclealkyl; and

R

4 is selected from chemical moieties (iv) through (vii) identified above.

As used herein, a "C3- 12 heterocycle" is a moiety that contains a ring made up of more than one kind of atom, and which contains 3 to 12 carbon atoms. In one embodiment, the C 312 heterocycle is selected from (but not limited to) pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl, and thianaphthyl.

A "C 4 1 6 heterocyclealkyl" is a moiety that contains a C 312 heterocycle linked to a C,alkyl, and which contains 4 to 16 carbon atoms. In one embodiment, the

C

4 6 heterocyclealkyl is a methylene furan having the following structure: A "substituted"

C

3 1 2 heterocycle or C 4 .,heterocyclealkyl is a

C

3 1 2 heterocycle or C 4 6 heterocyclealkyl having one or more hydrogens replaced with a substituent selected from halogen (including -Cl, -Br and -OH, -OR, -COOH, -COOR, -COR, -CONH,,

-NH

2 -NHR, -NRR, -SH, -SR, -SOOR, -SO 3 R and -SOR, where each occurrence of R is independently selected from an unsubstituted or substituted

C,-

8 alkyl, C 6 2 aryl, C,,,aralkyl,

C

3 ,heterocycle or C 41 6 heterocyclealkyl as defined above.

In structure above, R 6 is selected from hydrogen,

-CH

3

-CH

2

C,

6 H, -F and -CF, WO 97/09325 PCT/US96/14089 9 In one embodiment, the compounds of this invention have structure

(I)

above wherein

R

5 is the chemical moiety In this embodiment, the compounds disclosed herein have the following structures (II) and (III): O OR 7

R

4 a

R

6

NYN

R

2 a O OR 7 R4b R6

NRN

R2b

(II)

(III)

wherein R 2 a, R 2 b, R 4 a, R 4 b, R and R, are as defined above.

In a preferred embodiment, the compounds of this invention have structure (II) above, wherein R 2 a, R 4 a, R and R, are selected from the moieties identified in Table 1 below.

WO 97/09325 PCT/US96/1 4089 Table I Comp~ounds of Structure (11)

R

2 a 0 0 o CH 3 N

N

0

R

4 -Cl

-CF

3

-CH

3 -phenyl

-(CH

2 12

CH

3

-C

2

F

3 -a/

R

6

R

7 -H

-CH

3

-CF

3 -CH 2

CH

3

-CH

3

-H

CH

3 1 0 N, N fH 0 wherein X, Y and Z are the same or different, and independently selected from hydrogen, -OH, -OR, -COOH, -COOR, -COR -CONH 2

-NH

2 -NHR,

-NRR,

-N21 -SH, -SR, -SOOR,

-SO

3 R and -SOR, where each occurrence of R is independently selected from an unsubstituted or substituted

C

1 8 ,alkyl,

C

6 ,aryl,

C

7 1 aralkyl, C 3 2 heterocycle or C.

1 6 heterocyclealkyl.

In a further preferred embodiment, the compounds of this invention have structure (111) above, wherein

R

2 b, ROb, R 6 and R, are selected from the moieties identified in Table 2 below.

WO 97/09325 PCT/US96/14089 11 Table 2 Compounds of Structure (III)

R

2 b

-CI

-CF

3

-CH

3 -phenyl -(CH2)1-2CH3

-C

2

F

3 -ox S X "0X

R

4 b R6

-H

-CF

3

-CH

3 R7

-CH

3

-CH

2

CH

3

-H

0 H CSH, oN 0 H N 0

CH

3 I 0 N N CH3

SN

o wherein X, Y and Z are the same or different, and independently selected from hydrogen, -OH, -OR, -COOH, -COOR, -COR -CONH 2

-NH

2 -NHR, -NRR,

-NO

2 -SH, -SR, -SOOR, -SO 3 R and -SOR, where each occurrence of R is independently selected from an unsubstituted or substituted

C.

8 alkyl, C 6 2 aryl,

C

712 aralkyl, C3 12heterocycle or C4-, 6 heterocyclealkyl.

In another embodiment, the compounds of this invention have structure above wherein R 5 is the chemical moiety In this embodiment, the compounds disclosed herein have the following structures (IV) and WO 97/09325 PCTIUS96/14089 0

R

8

R

4 ,aR NyN o R 8

R

4 br

R

R2b (IV)

(V)

wherein R 2

R

2 b, R 4 a, R 4 b, R 6 and R, are as defined above.

In another embodiment, the compounds of this invention have structure above wherein R, is the chemical moiety (iii). In this embodiment, the compounds disclosed herein have the following structures (VI) and (VII): R4a;, R6 0,1

'Y

R

2 a

(VI)

R4b

R

6 0,1 N )-N R2b

(VII)

wherein R 2 a, R 2 b, R 4 ROb, R 6 and R 7 are as defined above.

In one embodiment, the compounds of this invention have structure (11) or (III) above and include (but are not limited to) the following: ethyl 1'aminocitraconamido))4rifluoromethylpyrim idin-carblate ethyl

I

5-acetyl-2-(N-(

I'-

aminocitraconamido)).4-trifluoromethylpyrimidine; ethyl 1'-amino-3ethyl I'-amino- 3 4 ,ethyl

I'

ethyl 4- 1'-amino-3 -hnlaemd)--rfurmehlyiiie5croyae ethyl 4- WO 97/09325 PCT/1JS96/1 4089 13 ethyl 'annctaoaio)4mthlyiiie5croyae ethyl 1'- 1'methyl 1'ammnocitraconamido))-4-(3'-pyridyl)pyrimidine.5-.carboxylate; diethyl

V-

ethyl '-amino-3'methyl

I'-

ethyl 2-[N- (1 ethyl 2- I'-amino-3 4 ethyl 2[-I-mnctaoaio)-yiiie5croyae ethyl N-(1' ethyl

I'-

ethyl 2-[N-acetyl.

'aioircnmd)--rpy-yiiie5croyae ethyl 2-[N-acetyl-N- (1 methyl 1'methyl 2-[N- (methyl)-N-( I'-amino-3 carboxylate; t-butyl-2- l'-aminocitraconamido)-4-trifluoromethylpyrimidinecarboxylate; methyl-2-[N-( carboxylate; methyl-2-[N-( l'-amninocitraconamido)-N..methyl]..4-tifluoromethy..

methyl 2- 1'-aminocitraconamido)]-4-.(2'.

ethyl 1'-aminocitraconamido)-N-.benzyrl]-4 ethyl 2- 1'-aminocitraconamido)-N.

2- 1'-aminocitraconamido)-4trifluoromethyl-pyrimidine-5-carboxylic acid; ethyl 2- I'-aminocitraconamido)] -4- (3 '-thienyl)pyrimidine-5-carboxylate; ethyl 2- 1'-aminocitraconamido)-N-methyl] -4- (3 'thienyl)pyrimidine-5carboxylate; ethyl 2- 1'-aminocitraconamido)]-4-(5 '-methyl- 2 -thienyl)pyrjmidine..s-carboxylate; ethyl 2- 1'-aminocitraconamido)]-4-.(2'..

ethyl 2- 1'-amninocitraconamido)-N-methyl]4-(5 methyl- 2 '.thienyl)pyIrimidine5carboxylate; ethyl 2- '-amino-3'-methylmaleimido)]- WO 97/09325 PCTIUS96/14089 14 4 2 '-thianaphthyl)pyridine-5carboxylate; methyl-2-[N-( I -alniinocitraconamido..N ethyl]- 4 -trifluoromethyl.-pyimidine5carboxylate; ethyl 1 -aminocitraconamido..

ethyl

I'-

am~inocitraconamidoN .{Nmetharoxylrboxamjdl)]romethl.imdne carboxylate; ethyl 1'-aminocitraconanido).N.( 1 -oxo-2-phenylethyl)-4ethyl 1'-amninocitraconamido)].4 ethyl 1'-aminocitraconamido)-N-.

ethyl

I'-

ethyl-2ethyl 1'-amninocitraconalnido)]-4. 2 ethyl ethyl 2l'-aminocitraconanidoNme thyl]4(2'-titazolyl yrimidi-cabxla ethyl l'-aminocitraconamido)].4-cyclopropylpyrimji ine-5carboylate and ethyl 2-[N- (1 In another embodiment, the compounds of this invention have structure (IV) or above and include (but are not limited to) the following: 5-benzoyl-2chloro- 4 -trifluoromethylpyimidine; 5-acetyl-2- I'-amninocitraconamnide)]-4 trifluoromethylpyimidine; 5-benzoyl-2- 1'-anlinocitraconarnido)]-4 trifluoromethylpyrimidine; 5-benzoyl-2-[N-( I'-aminocitraconamido)]-4 ethylpyrimidine; 5-benzoyl-2-[N-( 1'-aminocitraconamido-N-methyl].4ethylpyrimidine; and 5-butanoyl-2-[N-( 1'-aminocitraconamido..N-.methyl].4 ethylpyrimidine.

In yet another embodiment, the compounds of this invention have structures (VI) or (VII) above, and include (but are not limited to) the following: methylol-2- 1'-aminocitraconamido)] 4 -ethylpyrimidine; 5-methylol-2-[N-(

I'

aminocitraconamido)Nmethyl]-4ethylpyrimidine; 5-methoxymethane-2-[N.(

I'

aminocitraconamido)Nmethyl]A4ethylpyrrnidine; 5-methoxymethane-2-[N..(

I'-

aminocitraconamido)]-..trifluoromethylpyinidile; and 5-ethoxymethylcarbonate-2.

'aioircnmd)--tilooehlyiiie WO 97/09325 PCT/US96/1 4089 Preferred compounds of the invention are ethyl ethyl 1'aminophthalimido))4trifluoromethylprimidnecarblate 5-acetyl-2-(N( 1'ethyl 1'amino-3 ethyl 1'amino-3', ethyl 2- I'-aminocitraconamido)N methyl).4.trifluoromethyl ethyl 1'-amino-3 ethyl -amino-3', 4 '-dimethylmaeimido))2.tifluoromethypyimidincarboxylate; ethyl ethyl l'-aminocitraconamido))4pentafluoroethylpymidinecboxilte ethyl methyl 1'aminocitraconamido)).4-.(3 '-pyridyl)pyrimidine..s.carboxylate; diethyl

I

ethyl 1'ethyl 1'amnctaoand)Nmthl -typrmdn--carboxylate; methyl 1'ethyl ethyl

I'-

aiioircnmd)--5-ehl2-hey~yiiie5croyae ethyl

I'

and ethyl 1' aminocitraconamido))2(2thieny)pymde-5.cbxylt The compounds of this invention may be made by one skilled in organic synthesis by known techniques, as well as by the synthetic routes disclosed herein.

Referring to Figure 1, the compounds of this invention may be made from commercially available P-keto esters 1 by heating at elevated temperatures (75-1 I 0 0

C)

with a mixture of urea and triethylorthoformnate (or a substituted orthoformate) to provide ureido derivatives 2. Treatment of these intermediates with sodium alkoxides, such as sodium ethoxide in an alcoholic solvent at 35-1001C, gives 2hydroxypyrimidine esters 3 which, upon treatment with a chlorinating agent such as WO 97/09325 PCT/US96/14089 16 phosphorous oxychloride at elevated temperatures (75-120 0 yields 2chloropyrimidine esters 4.

Compound 4 may be reacted with various nucleophiles in an aprotic solvent at ambient temperature to provide derivatives 7. Compound 4 may also be converted to the carbonyl chloride 5 by treatment with base, such as hydroxide in water, followed by a chlorinating agent, such as oxalyl chloride in methylene chloride.

Compound 5 can be treated with an organometallic, such as methyl magnesium bromide in a solvent such as THF or ether at -35 0 C to -65 0 C, to give ketone 9. This ketone may be treated with various nucleophiles in an aprotic solvent and at ambient temperature to provide compound Alternatively, compound 3 may be converted to the hydroxy carboxylic acid_6 by treatment with a strong base, such as sodium hydroxide, or strong acid, such as HC1, at elevated temperature (70-110°C). The hydroxy carboxylic acids may be converted to the chloro carbonyl chloride with thionyl chloride and/or phosphorous oxychloride.

Compound 4 can also be treated with hydrazine at ambient temperature in a solvent, such as THF, with pyridine as a catalyst to provide the intermediates of structure 8. These hydrazino derivatives can be reacted with cyclic anhydrides, such as citraconic anhydride, in a solvent, such as chloroform, at elevated temperatures (35-65 0 C) to provide compounds of structure 11. Subsequent treatment of 11 with a strong base, such as sodium hydride, in an aprotic solvent, such as THF, at ambient temperature followed by an alkyl iodide, such as methyl iodide, provides the alkylated derivatives of structure 12.

Compounds of structures (VI) and (VII) may be prepared by reducing any of the compounds in Figure 1 so as to convert a carboxylate group to a methylol (-CHOH) group. Lithium aluminum hydride is a suitable reducing agent. In any event, the methylol group may, if desired, be converted to -CH 2 OR, by standard alkylation chemistry using a strong base and a nucleophile).

Referring to Figure 2, an alternative synthetic procedure is disclosed. In this procedure, commercially available diethyl ethoxymethylenemalonate 13 is treated WO 97/09325 PCT/US96/14089 17 with an amidine, such as trifluoromethylamidine, in a protic solvent, such as ethanol, in the presence of an alkoxide, such as NaOEt, at elevated temperatures (75-110°C) to give the hydroxy pyrimidine ester 14. Chlorination with a chlorinating agent, such as POCI3 or thionyl chloride, yields the chloroester derivative 15 which can be treated with various amines at ambient temperature in an aprotic solvent, such as THF, to provide the substituted pyrimidines 16. Derivative 15 may also be treated with hydrazine in a solvent such as THF in the presence of pyridine to give the hydrazino intermediates 17.

Treatment of 17 with various cyclic anhydrides, such as citraconic anhydride, in a solvent, such as chloroform, at elevated temperatures (34-65 0 C) provide compounds of structure 18. Alkylation of these compounds with a hydride, such as sodium hydride, followed by an alkyl halide in an aprotic solvent, such as THF, gives the alkylated derivatives of structure 19.

Again, compounds of structures (VI) and (VII) may be prepared from the corresponding carboxylate compounds as discussed above in connection with Figure 1.

Compounds of the invention wherein R, is may be prepared from the corresponding compound wherein R is hydrogen (as prepared according to either of Figure 1 or 2) using standard acylation chemistry. For example, a compound having R, equal to hydrogen may be treated with a strong base NaH), followed by an acylating agent Once synthesized, the compounds of this invention may be formulated for administration to a warm-blooded animal by a variety of techniques known to those skilled in the art. In one embodiment, the compound is in the form of a pharmaceutical composition for prophylactic or therapeutic use, and which contains at least one compound of this invention in combination with a pharmaceutically acceptable carrier or diluent. The compound is present in the composition in an amount which, upon administration to the animal, is effective in preventing or treating the condition of interest. Preferably, the composition includes a compound of this invention in an amount ranging from 0.01 mg to 250 mg per dosage, depending upon the route of administration, and more preferably from 1 mg to 60 mg. Appropriate concentrations, WO 97/09325 PCT/US96/14089 18 dosages and modes of administration may be readily determined by one skilled in the art.

Suitable carriers or diluents are familiar to those skilled in the formulation field. For compositions formulated as liquid solutions, acceptable carrier or diluents include saline and sterile water, and may optionally include antioxidants, buffers, bacteriostats and other common additives. The compositions of this invention may also be formulated as pills, capsules, granules or tablets which contain, in addition to the compound of this invention, diluents, dispersing and surface active agents, binders and lubricants. One skilled in the art may further formulate the compounds of this invention in any appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1990 (incorporated herein by reference).

In another embodiment, the present invention provides methods for preventing or treating a variety of conditions. Such methods include administering a compound of this invention to a warm-blooded animal in need thereof in an amount sufficient to prevent or treat the condition. Such methods include systemic administration of a compound of this invention, preferably in the form of a composition as disclosed above. As used herein, systemic administration includes oral and parental methods of administration. For oral administration, suitable pharmaceutical compositions include powders, granules, pills, tablets and capsules, as well as liquids, syrups, suspensions and emulsions. These compositions may also include flavorants, preservatives, suspending, thickening and emulsifying agents, and other pharmaceutically acceptable additives. For parental administration, the compounds of the present invention may be prepared in aqueous injectable solutions which may contain, in addition to the compound of this invention, buffers, antioxidants.

bacteriostats and other additives commonly employed in such solutions.

As mentioned above, compounds of the present invention can be used to prevent or treat a wide variety of disorders, diseases and/or illnesses. In particular, the compounds may be administered to a warm-blooded animal for prevention or treatment of rheumatoid arthritis, osteoarthritis, tissue and/or organ transplant rejection, sepsis, WO 97/09325 PCT/US96/14089 19 ARDS, asthma, trauma, oxidative stress, cell death, irradiation damage, ischemia, reperfusion, cancer, viral infection, and autoimmune diseases such as psoriasis, inflammatory bowel disease, glomerulonephritis, lupus, uveitis and chronic hepatitis.

Compounds of this invention may be screened by known and accepted techniques for their ability to function as prophylactically and/or therapeutically active agents. For example, the compounds may be evaluated in in vitro and/or in vivo assays indicative of the compound's anti-inflammatory and immunosuppressive properties. To this end, such compounds may first be evaluated in a number of cell-based assays which determine the ability of a compound to prevent activation of NFKB and AP-1 (see Example 126). Next, the compound's ability to attenuate cytokine levels (such as IL-2 and IL-8), which are known to be elevated in certain disease states, may be determined (see Example 127). The compounds may then be evaluated in an appropriate animal model, including rodent models of inflammation and immunosuppression (see Example 128).

It should be recognized that, for example, in the case of immunosuppressive drugs and other agents which have utility for the treatment of rheumatoid arthritis numerous studies have been performed directed to the activity of such drugs. To this end, cyclosporin A has been used in clinical trials since the late 1970's as a second-line drug, and is recommended to be used only in patients with active RA. Thus, Experiment 128 may be performed utilizing cyclosporin A as a positive control. A recent review of such immunosuppressive drugs, including relevant assays for the same, is presented by R.P. Carlson in Exp. Opin. Invest. Drugs 4(9):853- 859, 1995 (incorporated herein by reference in its entirety, including cited references).

The following examples are presented for purpose of illustration, not limitation.

EXAMPLES

To summarize the examples that follow, Examples 1-124 disclose the synthesis of representative compounds of this invention, as well as intermediates thereto; Example 125 discloses the synthesis of representative compounds by WO 97/09325 PCT/US96/14089 combinational chemistry techniques; Examples 126-127 disclose the ability of representative compounds of this invention to inhibit NFcB, AP-1 and cytokines; and Example 128 discloses assays for evaluating activity of representative compounds of this invention in both graft versus host disease and contact sensitivity models.

Example 1 2-CHLORO-5-[3',5'-BIS(TRIFLUOROMETHYL)PHENACYL]- 4

-TRIFLUOROMETHYLPYRIMIDINE

To magnesium turnings (0.026 g, 1.06 mmol) in Et2O (15 mL) was added a solution of 3,5-bistrifluoromethyl iodobenzene (0.300 g; 0.882 mmol) in Et 2

O

mL). The reaction was refluxed under an atmosphere ofN 2 for 2 h and then cooled to 0°C. A solution of 2 -chloro-4-trifluoromethylpyrimidine-5-carbonyl chloride (0.205 g, 0.838 mmol) in Et2O (5 mL) was added dropwise via syringe. After stirring 1 hour at 0°C, water (15 mL) was added and the mixture extracted with Et20 (2 X 20 mL).

The organic layer was washed with brine (15 mL), dried over MgSO 4 filtered and concentrated. The residue was chromatographed (SiO 2 hexanes/EtOAc 8:1) to give the title compound (0.078 g, 21% yield) as an oil; 'H NMR (CDCl 3 8 8.90 1H), 8.21 (s, 1H), 8.19 2H).

Example 2 5-BENZOYL-2-CHLORO-4-TRIFLUOROMETHYLPYRIMIDINE The title compound was prepared as described in Example 1, but employing phenyl magnesium bromide (0.23 mL, 0.69 mmol) and the acid chloride (0.17 g, 0.69 mmol), resulting in a yield of 30%; 'H NMR (CDC1 3 8 8.81 1H), 7.4- 7.8 WO 97/09325 PCT/US96/14089 21 Example 3 ETHYL UREIDOMETHYLENE

ACETOACETATE

A mixture of ethyl acetoacetate (200 g, 1.54 mole), urea (105 g, 1.54 mole) and triethyl orthoformate (228 g, 1.54 mole) was heated at 140 0 C under N 2 for 22 h. The reaction mixture was cooled and filtered to provide the title compound in a 51% yield (156 m.p. 173-174C.

Example 4 ETHYL UREIDOMETHYLENE

BENZOYLACETATE

The title compound was prepared as described in Example 3, but employing ethyl benzoylacetate (30.0 g, 156 mmol), resulting in a yield of 21% (12g); m.p. 124-126 0

C.

Example ETHYL 2-HYDROXY-4-METHYLPYRIMIDINE-5-CARBOXYLATE A solution of ethyl ureidomethylene acetoacetate (50 g, 250 mmol) NaOEt (22.1 g, 325 mmol) in EtOH (500 mL) was stirred at room temperature under N 2 for 3 days. The resulting solid was filtered and dried to yield the title compound as a sodium salt in a yield of 88% (45 m.p. >220 0 C (dec.).

Example 6 ETHYL 2 The title compound was prepared as described in Example 5, but employing ethyl ureidomethylene benzoyl acetate (12 g, 45 mmol), resulting in a yield of 15% (6 m.p. >260 0 C, (dec.).

WO 97/09325 PCT/US96/14089 22 Example 7 ETHYL 2-HOO4MTYPRMDN--ABXLT A solution of ethyl 2 -hydroxy-4methylpyrimidine5-carboxylate (5 g, 27.5 mmol) and POC1 3 (84 g, 550 mmol) was heated at reflux under N 2 for 1 h. The reaction was cooled and concentrated. The residue was partitioned between

CHC

3 and

H

2 0 and the organic layer was dried (Na 2 SOA) filtered, and concentrated to yield the title compound in a yield of 27% (1.5 'H NMR (CDCI 3 8 9.04 IlH), 4.42 (q, 2H), 2.85 3H), 1.43 3H).

Example 8 ETHYL 2 The title compound was prepared as described in Example 7, but employing 2-yrx--hnlyimdn--abxlt (6 g, 25 mmol) to give the title compound (5.5 g, m.p. 45-47"C.

Example 9 2 -CHLORo-4-METHYLPYRIMIDINE5-CARBOXYLIC

ACID

A solution of ethyl 2 -chloro-4-methylpyrimidine-.5-carboxylate (1.0 g, mmol), NaOH (0.24 g, 6 mmol) in H 2 0 (30 mL) was stirred at room temperature for 3 h. The solution was acidified with 6N HCl and the resulting solid was filtered and dried to give the title compound (0.67 g 'H1 NMR (DMSO-d 6 6 9.01 1H), 2.75 (s, 3H).

Example 2 -CHLORo-4-PHENYLPYRIMIDINE.5 -CARBOXYLIC

ACID

The title compound was prepared as described in Example 9, but employing 2-hoo4peyprmdn--abxlt (4.5 g, 17 mmol), resulting in a yield of 87% (3.9 m.p. 105-1 101C.

WO 97/09325 PCT/U596/14089 23 Example 11I 2 -CHLORo-4-METHYLPYRIMIDINE.5-.CARBONYL

CHLORIDE

A solution of 2 -chloro- 4 -methylpyrimidine-5-carboxylic acid (0.81 g, 4.70 mmol), oxalyl chloride (0.89 g, 7.05 mmol), DMF (2 drops) in CH 2 C1 2 (23 mL) was stirred at room temperature under N 2 for 4 h. The solution was concentrated and distilled to give the title compound (0.55 g, 61 h.p. 90-100 0 1.3 mm/Hg; 'H NMR (CDCl 3 6 d 9.02 1H), 2.74 3H).

Example 12 2 -CHLORo-4-PHENYLPYRIMIDINE.5-.CARBONYL

CHLORIDE

The compound was prepared as described above in Example 11, but employing 2 -chloro- 4 -phenylpyrimidine5carboxylic acid (3.8 g, 14 mmol), resulting in a yield of 53%; m.p. 42'C.

Example 13 2 The compound was prepared as described in the literature (see Arukwe, J Undheim, K Acta Cliemica Scand. B40:764, 1986).

Example 14 ETHYL ETHOXYMETHYLENE-4,4,4.TRIFLUOROACETOACETATE A solution of ethyl 4 4 4 -trifluoroacetoacetate (46 g, 0.25 mol) triethyl orthoformate (74 g, 0.50 mol) and Ac 2 O (77 g, 0.75 mol) was heated at 120-140'C for 7 h. The mixture was concentrated and distilled to give the title compound in a 98% yield (58.6 b.p. 80-90'C, 1.5 mm/Hg.

WO 97/09325 PCTIUS9614089 24 ExaMpie ETHYL 2 A solution of diethyl ethoxymethylenemalonate (35.0 g, 162 mmol), trifluoroacetamidine (18 g, 162 mmol) and NaOEt (11. 0 g, 162 mmol) in EtOH (200 mL) was heated at reflux for 6 h. The reaction mixture was concentrated and H 2 0 (48 mL) was added. The resulting solid was filtered, washed with Et 2 O (300 mL) and H 2 0 (200 mL), and dried to give the title compound (21 g, 50% yield); m.p. >220'C (dec.); 'H NMR (DMSO-d 6 8 8.38, 4.16 2H), 1.25 3H).

Example 16 2 -TRIFLUROROMETHYL4-CHLQROPYRIMIDINE5CARBONYL

CHLORIDE

A solution of ethyl 2 carboxylate (5.00 g, 19.4 mmol) and NaOH (0.93 g, 23.3 mnmol) in H 2 0 (20 mnL) was stirred at 60'C for 15 h. The reaction was acidified (co ncentrated HQl and concentrated until a solid began to form. The solid was filtered and dried to give 2acid (2.1 g, 53% yield); 'H NMR (DMSO-d 6 8 8.83 I H).

A solution of 2 -trifluoromethy1-4-hydroxypyrimidine-5.carboxylic acid g, 10.4 mmnol), POCd 3 (32 g, 212 mmol) and SOC1 2 (25 g, 212 mmol) was heat ed at reflux for 4 days. The reaction was concentrated and distilled 90-95'C, mm/Hg) to provide the title compound (2.1 g, 8 1% yield), 'H NMR (CDCl 3 8 9.45 (s, 1H).

Example 17 2 -CHLORo-4-PENTAFLUOROETHYLPYRIMIDNE5CARBONYTL

CHLORIDE

A solution of ethyl 2 carboxylate (4.0 g, 13 mmol) and NaOH (1.60 g, 39 mmol) in EtOH (20 mL) and H 2 0 mL) was heated at reflux for I h. The solution was cooled and acidified (concentrated HQl. The resulting solid was filtered and dried to provide 2-hydroxy-4- WO 97/09325 PCTIUS96/1 4089 acid (3.3 g, 98% yield); 'H NMR (DMSO-d 6 8 9.90 (bs, I 8.43 I H).

A solution of 2-yrx--etfurehlyiiie5croyi acid (3.33 g, 12.9 mmol) in SOC1 2 (27.7 g, 233 mmol) was heated at reflux for 0.5 h. Then POCd 3 (35.6 g, 233 mmol) was added to the reaction mixture and heating continued for 36 h. The reaction mixture was then concentrated and distilled 80-85'C,

I

mm/Hg) to give the title compound (1.2 g, 35% yield); 'H NMR (DMSO-d 6 )569.18 (s,

IL)

Example 18 ETHYL 4 A solution of ethyl 4 -chloro- 2 (0.20 g, 0.79 mmol), hydrazine (0.18 g, 6.0 mmol) and THF was stirred for 1 h at room temperature. The solution was filtered and dried to give the title compound in a 96% yield; 'H NMR (CDCl 3 69.26 (bs, I1H), 8.90 I1H), 4.40 2H), 4.24 (bs, 1.41 3H).

Example 19 ETHYL 2 The title compound was prepared as described in Example 18, but employing ethyl- 2 -chloro-4-trifluoromethylpyrimidine-5carboxylate (0.20 g, 0.79 mmol), resulting in a yield of 91% (0.18 m.p. 89-90'C.

Example ETHYL 2- I'-AMINOCITRACONAMIDO)] 4

-TRIFLUOROMETHYL-

A solution of ethyl 2 -hydrazino-4-trifluoromethylpyrimidines...

carboxylate 18 g, 0.72 mmol) and citraconic anhydride (0.08 ml, 0.94 mmol) in CHC1 3 (10 ml) was refluxed for 0.5 h. The solution was cooled, concentrated and WO 97/09325 PCTIUS96/I 4089 26 chromatographed (Si0 2 hexanes/EtOAc) to give the title compound 10 g, 3 9% yield); 'H NMR (CDCI 3 )869.94 1H), 7.72 1H), 6.53 IH), 4.41 2H), 2.19 (s, 311I), 1.36 3H).

ExaMple 21 ETHYL l'-AMINOCITRACONAMIDO)].2-TRIFLUOROMETHYL- The title compound was prepared as described in Example 20, but employing ethyl 4-yrzn--rfurmtyprmdn--abxlt (0.19 g, 0.76 mniol), resulting in an 80% yield (0.21 m.p. 85-86 0

C.

ExaMple 22 ETHYL '-AMINOPHTHALIMIDO)]-4TRIFLUOROMETHYL- A solution of ethyl 2 -chloro-4-trifluoromethyl..spyrimidine ester (0.25 g, 1.0 mmol), N-aminophthalimide 17 g, 1.0 mmol) and pyridine (0.09 ml, 1.0 mmol) in THF was heated at 60'C for 5 h and then concentrated. The residue was chromatographed (SiO 2 hexanes/EtOAc, 1:1) to give the title compound (0.07 g, 17% yield); m.p. 46-48 'C.

Exam]2le 23 5-ACETYL-2-[N-( 1'-AMINOCITRACONAMIDO)]- 4

-TRIFLUOROMETHYLPYRIMIDINE

To a solution of 2 -chloro- 4 chloride (0.50 g, 2.0 mmol) in THF at -78'C under N, was added MeMgBr (0.75 ml, 2.3 mmol). The reaction was stirred 0.75 h, quenched with H2,O(Imi) and diluted with EtOAc (30 ml). The organic layer was washed with H 2 0, brine and then dried over MgSO,. The residue was chromatographed (SiC 2 hexanes/EtOAc, 2:1) to provide the 5-ctl2clr--rfurmtyprmdn (0.20 g) in 43% yield. The title WO 97/09325 PCT/US96/14089 27 compound was then prepared as described for ethyl 2-[N-(l'-aminocitraconamide)]-4of Example 20, resulting in a 29% yield (0.08 m.p. 61-62°C.

Example 24 ETHYL UREIDOMETHYLENE

PROPIONOYLACETATE

The title compound was prepared as described in Example 3, but employing ethyl propionylacetate (5.15 g, 35.7 mmol), resulting in a yield 43% (3.29 m.p. 148-150 0

C.

Example ETHYL UREIDOMETHYLENE

BUTYRYLACETATE

The title compound was prepared as described in Example 3, but employing ethyl butyrylacetate (25 g, 158 mmol), resulting in a yield of 47% (17g); m.p. 145-147°C.

Example 26 ETHYL UREIDOMETHYLENE 2

-THIENYLACETATE

The title compound was prepared as described in Example 3, but employing ethyl 2 -thienoylacetate (7.22 g, 36.4 mmol), resulting in a yield of 41% m.p. 149-150 0

C.

Example 27 ETHYL 2 The title compound was prepared as described in Example 5, but employing ethyl ureidomethylene ethanoylacetate (2.5 g, 11.7 mmol), resulting in a yield of 52% (1.2 'H NMR (DMSO-d 6 6 8.05 1H), 4.24 2H), 2.87 2H), 1.29 3H).

WO 97/09325 PCTIUS96/1 4089 28 Example 28 ETHYL 2-HYDROXY 4 The title compound was prepared as described in Example 5, but employing ethyl ureidomethylene propionoylacetate (10.96 g, 48 mmol), resulting in a yield of 73% (7.3 'H1 NMR (CDCl 3 8 8.2 11H), 4.35 2H), 3.0 2H), 1.75 (in, 2H), 1.37 3H), 1.0 3H).

Example 29 ETHYL 2-YRX--2-HEY)PRM~N--ABXLT The title compound was prepared as described in Example 5, but employing ethyl ureidomethylene 2 -thiophenoylacetate (4.0 g, 14.9 nimol), resulting in a yield of 79% (2.9 m.p. 144-146'C.

Example ETHYL 2 The title compound was prepared as described in Example 7, but employing ethyl 2 -hydroxy-4-ethylpyrimidine.s.cabxylate (1.2 g, 6.1 nimol), resulting in a yield of 77% (1.0 GU/MS calcd for C 9

H

11

IN

2 0 2 C1 214, found 214.

Example 31 ETHYL 2 The title compound was prepared as described in Example 7, but employing ethyl 2-yrx--rplyimdn--abxlt (1.05 g, 5 mmol), resulting in a yield of 79% (0.9 GC/MS calcd for CJQH 1 3

N

2

O

2 CI 228, found 228.

WO 97/09325 PCT/US96/14089 29 ExaMple 32 ETHYL 2-HOo4('TINLPRMDN--ABXLT The title compound was prepared as described in Example 7, but employing ethyl 2 -hydroxy- 4 -(2'-thienyl)pyrimidine.5-.carboxylate (1.0 g, 4.0 mmol), resulting in a yield of 19% GCIMS calcd for CjIHqN 2 O.,SCI 268, found 268.

Example 33 ETHYL 2 The title compound was prepared as described in Example 18, but employing ethyl 2 -chloro- 4 -ethylpyrimidine5carboxylate (1 g, 4.7 mmol), resulting in a yield of 41% (0.4 GC/MS calcd for C 9 11 14

N

4 0 2 210, found 210.

Example 34 ETHYL 2-HYDRAZINO-4-PROPYLPYRIMIDINE..5.CARBOXYLATE The title compound was prepared as described in Example 18, but employing ethyl 2 -chloro- 4 -propylpyrimidine.s..carboxylate (0.9 g, 3.9 nimol), resulting in a yield of 97% (0.85 GC/M4S calcd for C 10

H

16

N

4 0 2 224, found 224.

Example ETHYL 2-YRZ~--2-HEY)PRMDN--ABXLT The title compound was prepared as described in Example 18, but employing ethyl 2 -chloro- 4 2 '-thienyl)pyrimidine...scarboxylate (0.2 g, 0.7 mmol), resulting in a yield of 92% (0.17 GC/MS calcd for CI 1

H

12

N

4 0 2 S 264, found 264.

WO 97/09325 PCT/US96/14089 Example 36 ETHYL '-AMIOCITRACONAMIDO)]-4-ETHYLPYRIMIDINE-5-CAR13XYLAT The title compound was prepared as described in Example 20, but employing ethyl 2 -hydrazino-4-ethylpyrimidine-5-carboxylate (0.4 g, 1.9 mmol), resulting in a yield of 32% (0.13 'H NMR (CDCI 3 5 8.84 IH), 6.51 1H), 5.94 1H), 4.32 2H), 3.05 2H), 2.18 3H), 1.35 6H).

Example 37 ETHYL The title compound was prepared as described in Example 20, but employing ethyl 2 -hydrazino-4-propylpyrimidine-5-carboxylate (0.77 g, 3.4 miol), resulting in a yield of 73% (0.7 m.p. 103-105C.

Example 38 ETHYL '-AMINOCITRACONAMIDO)]-4-(2'-THIENYL)PYRIMIDINE-5-CAROXYLATE The title compound was prepared as described in Example 20, but employing ethyl 2 -hydrazino-4-(2'-thienyl)pyrimidine-5-cboxylate (0.17 g, 0.64 imol), resulting in a yield of 55% (0.127 m.p. 123-126 0

C.

Example 39 5-AMIDo-2-[N-(I

'-AMINOCITRACONAMIDO)]-

4

-TRIFLUOROMETHYLPYRIMDINE

To a solution of 2 -chloro- 4 chloride (0.50 g, 2.0 mmol) in THF at 0 0 C under N, was added 2M NH 3 /MeOH (1.0 ml, 2.0 mmol). The reaction was stirred 5 minutes, diluted with EtOAc (10 ml), filtered and concentrated. Chromatography (SiO 2 hexanes/EtOAc, 4:1) provided the 5-amido-2chloro- 4 -trifluoromethylpyrimidine (0.20 g) in 44% yield. The title compound was then prepared as described for ethyl 2 -[N-(l'-aminocitraconamide)]-4resulting in a 15% yield (0.04 'H NMR WO 97/09325 WO 9709325PCT/US96/1 4089 31

(CDCI

3 8 8.69 1H), 8.30 1H), 6.52 6.51 11H), 6.28 11H), 2.19 (s, 3H).

Example ETHYL 2-IIN-( I'-AMINo-3'-PHENYLMALEIMIDO)]4.TRIFLUOROMETHYL- The title compound was prepared as described for ethyl 1'but employing 3phenylmaleic anhydride (0.21 g, 1.2 mmol), resulting in a 74% yield (0.18 m.p.

52-53 0

C.

Example 41 ETHYL I'-AMINo-3 4 t -DIMETHYLMALEIMIDO)].4TRIFLUOROMETHYL.

The title compound was prepared as described for ethyl

F-

amnctaoaio]4tiloomtyprmdn--abxlt but employing 3,4dimethylmaleic anhydride (0.08 g, 0.63 mmol), resulting in a 47% yield (0.10 m.p.

110-111loc.

Example 42 ETHYL '-AMINOCITRACONAMIDo)-N-METHYL]-4TRIFLUOROMETHYL- To a solution of ethyl 2 -IIN-(l'-aminocitraconamido)]4-trifluoromethyl.

10 g, 0.29 mmol) in TI-F at 0 0 C under nitrogen was added NaH (0.01 g, 0.44 mmol). After 5 minutes, Mel (0.10 ml, 1.6 mmol) was added and the reaction was allowed to warm to room temperature. After stirring 0.5 h at room temperature, the reaction was diluted with EtOAc (15 mL), washed with H 2 0 and brine.

The organic layer was dried over MgSO,, filtered and concentrated. The residue was chromatographed (Si0 2 hexanes/EtOAc 8:1) to give the title compound (0.05 g, WO 97/09325 WO 9709325PCT/US96/1 4089 32 yield); 1H NMR (CDCl 3 69.05 11H), 6 .48 lH), 4.37 211), 3.61 2.17 (s, 1.35 31-1).

Example 43 ETHYL 1'-AMINo-3'-PHENYLMALEIMIDO)] 2

-TRIFLUOROMETHYL-

The title compound was prepared as described in Example 20, but employing a solution of ethyl 4 -hydrazino- 2 (0.09 g, 0.36 mmol) and 3-phenylmaleic anhydride (0.13 g, 0.72 mmol) resulting in a 69% yield 10 m.p. 179-180'C.

Example 44 ETHYL 1 -AMINo-3', 4 '-DIMETHYLMALEIMIDO)].2-TRIFLUOROMETHYL.

The title compound was prepared as described in Example 20, but employing a solution of ethyl 4 -hydrazino- 2 (0.09 g, 0.36 mmol) and 3,4-dimethylmaleic anhydride (0.09 g, 0.72 mmol) resulting in a 89% yield 12 m.p. 11I6-1 17*C.

Example ETHYL 2 -HYDRAZINo-4-METHYLPYRIMID1NE-.5..CARBOXYLATE The title compound was prepared as described in Example 18, but employing a solution of ethyl 2 -chloro- 4 -methylpyrimidine-5-carboxylate (0.08 g, 0.39 mmol) and hydrazine (0.06 g, 2.0 mmol) in THIF (7.8 mL) resulting in a 93% yield (0.07 'H NMR (CDCl 3 6 8.86 1H), 6.64 (bs. 1H), 4.33 2H), 2.70 1.38 31-).

WO 97/09325 WO 9709325PCT/US96/14089 33 Example 46 ETHYL I'-AMINOCITRACONAMIDO)]-4-METHYLPYRIMIDINE.

The title compound was prepared as described in Example 20, but employing a solution of ethyl 2 -hydrazino-4-methylpyrimidine-5-carboxylate (0.07 g, 0.36 mmol) and citraconic anhydride (0.08 g, 0.72 mrnol) resulting in a 52% yield (0.06 g m.p. 49-50*C.

Example 47 ETHYL 2 -HYDRAZINo-4-PENTAFLUOROETHYLPYRIMIDINE.

The title compound was prepared as described in Example 18, but employing a solution of ethyl 2 -chloro- 4 (0.30 g, 0.99 mmol) and hydrazine (0.16 g, 5.0 mmol) in THF (20 mL) resulting in a 95% yield (0.28 'H NMR (CDCl 3 8 8.86 (bs, 1H), 7.12 (bs, 1H), 4.37 2H), 1.72 (bs, 2H), 1.38 3H).

Example 48 ETHYL 1'-AMINOCITRACQNAMIDO)]-4-PENTAFLUOROETHYLPYRIMIDINE.

The title compound was prepared as described in Example 20, but employing a solution of ethyl 2 (0.28 g, 0.93 mmol) and citraconic anhydride (0.13 g, 1.1 mmol) resulting in a 68% yield (0.25 m.p. 73-74'C.

Example 49 ETHYL 2 The title compound was prepared as described in Example 18, but employing a solution of ethyl 2-chloro-4-phenylpyrimidine-5-carboxylate (0.09 g, 0.34 WO 97/09325 WO 9709325PCTIUS96/14089 34 mmol) and hydrazine (0.06 g, 1.7 mmol) in THF resulting in a 91% yield (0.08 m.p.

74-75 0

C.

Example ETHYL l'-AMIN0CITRACONAMIDO)]-4PHENYLPYRIMIDNE..s.CARBOXYLATE The title compound was prepared as described in Example 20, but employing a solution of ethyl 2 -hydrazino- 4 -phenylpyrimidine-5car.boxylate (0.08 g, 0.31 mmol) and citraconic anhydride (0.04 g, 0.37 minol) resulting in a 64% yield (0.07 m.p. 165-167C.

Example 51 ETHYL 2 -HYDRAZINo- 4 The title compound was prepared as described in Example 18, but employing a solution of ethyl 2 -chloro- 4 -benzylpyrimidine-5-carboxylate (0.34 g, 1.2 mmol) and hydrazine (0.2 g, 6.1 mmol) in THF resulting in a 99% yield (0.33 g, oil); GCUMS, 272(M-).

Example 52 ETHYL The title compound was prepared as described in Example 20, but employing a solution of ethyl 2 (0.34 g, 1.2 mmol) and citraconic anhydride (0.22 g, 2.0 mmnol) resulting in a 3 7% yield 15 g) of the title compound; m.p. 34-36'C.

Example 53 METHYL 2 -HYDRoxy-4-(3'-PYRIDYL)PYRIMIDINE..5.CARBOXYLATE The title compound was prepared as described in Example 5, but employing a solution of methyl ureidomethylene nicotinoyl acetate (8.4 g, 34 rnmol) Na WO 97/09325 PCT/US96/14089 g, 44 mmol) in EtOH (200 mL) resulting in a 52% yield (4.4 NMR (DMSOd 6 6 8.59 11H), 8.51 (in, 2H), 7.72 (in, 1H), 7.36 (in, 3.51 3H).

Exam.le 54 METHYL 2-CHLORo-4-(3 The title compound was prepared as described in Example 7, but employing a solution of methyl 2 -hydroxy- 4 3 '-pyridy)pyrimidine5carboxylate (4.42 18 mmol) and POC1 3 (53.6 g, 350 inmol) resulting in a 25% yield (1.1I 'H NMR (DMSO-d 6 8 9.11 1H), 8.77 (in, 2H), 8.03 (in, 7.45 (in, lH), 3.85 3H).

Example METHYL 2-HYDRAZINo-4-(3 The title compound was prepared as described in Example 18, but employing a solution of methyl 2 -chloro- 4 3 'pyridyl)pyrimidine5carboxylate g, 0.42 inmol) and hydrazine (67 mng, 2.1 mmol) resulting in a 97% yield 10 'H NMR (DMSO-d 6 8 9.09 1H), 8.76 11H), 8.69 8.35 (bs, IH), 7.89 (d, I1H), 7.42 (mn, I1H), 3.76 2H), 2.19(bs, 2H).

Example 56 METHYL '-AMINOCITRACONAMIDO)]-4(3'.PYRIDYL).

The title compound was prepared as described in Example 20, but employing a solution of methyl 2 -hydrazino- 4 (0.1 g, 0.39 inmol) and citraconic anhydride (0.04 g, 0.39 mniol) resulting in a 42% yield (0.06 m.p. 93-94'C.

WO 97/09325 PCT/US96/14089 36 Example 57 ETHYL 2 A solution of benzyl acetoacetate (7.0 g, 34 mmol) and N,Ndimethylformamide dimethyl acetal (4.0 g, 34 mmol) was stirred at room temperature for 0.25 h. The solution was concentrated and treated with urea (2.2 g, 37 mmol) and Na (1.2 g, 51 mmol) in EtOH (100 mL). The mixture was heated at reflux for 18 h, concentrated, acidified and purified by chromatography (SiO 2 to yield the desired product (2.6 g, m.p. 57-61 0

C.

Example 58 ETHYL 2 The title compound was prepared as described in Example 7, but employing a solution of ethyl 2 -hydroxy-4-benzylpyrimidine-5-carboxylate (1.1 g, 4.3 mmol) and POC1 3 (16 g, 107 mmol) resulting in a 37% yield (0.44 'H NMR (DMSO-d 6 6 9.00 1H), 7.6-7.4 5H), 4.55 2H), 4.25 2H), 1.14 3H).

Example 59 DIETHYL UREIDOMETHYLENE

OXALACETATE

A solution of diethyl oxalacetate (5.7 g, 30.4 mmol), triethyl orthoformate (8.8 g, 30 mmol) and urea (2.0 g, 30 mmol) was heated at reflux for 1.5 h.

The product was filtered, washed with water and ether to give (3.5 g, 45%) of the title compound; 'H NMR (DMSO-d 6 8 11.29 and 10.81 (dd, 1H), 8.6-7.4 3H), 4.2 (m, 4H), 1.24 6H).

Example METHYL UREIDOMETHYLENE NICOTINOYL ACETATE The title compound was prepared as described in Example 3, but employing a solution of methyl nicotinoyl acetate (10 g, 56 mmol), triethyl WO 97/09325 WO 9709325PCTIUS96/14089 37 orthoformate (8.3 g, 56 mmol) and urea (3.4 g, 56 mmol) resulting in a 60% yield (8.4 'H NMR (DMSO-d 6 6 10.85 and 10.36 (dd, lH), 8.8-7.2 (in, 7H), 3.58 3H).

Example 61 DIETHYL 2 A solution of diethyl ureidomethylene oxalacetate (18.3 g, 71 inmol) in xylene (95 ml) was heated at reflux for 18 h. The reaction was cooled and the resulting solid was filtered. The solid was recrystallized from EtOAc to give the desired compound (8.2 g, 'H NMR (DMSO-d 6 8 8.61 1H), 4.25 (mn, 4H), 1.27 (in, 6H).

Example 62 DIETHYL 2 The title compound was prepared as described in Example 7, but employing a solution of diethyl 2 -hydroxypyrimidine-4,5-dicarboxylate (1.0 g, 4.2 mmol) and POC1 3 (7.7 g, 50 inmol) resulting in a 30% yield (0.32 'H NMR (CDC1 3 6 9.09 1H), 4.35 (mn, 4H), 1.30 (in, 6H).

Example 63 DIETHYL 2 The title compound was prepared as described in Example 18, but employing a solution of diethyl 2 -chloroypyrimidine-4,5-dicarboxylate (0.21 g, 0.83 minol) and hydrazine 13 g, 4.2 inmol) in THF (10 mL) resulting in 96% yield (0.20 'H NMR (CDCI 3 8 8.93 (bs, IH), 8.34 (bs, IH), 4.40 (mn, 4H), 4.18 (bs, 2H), 1.35 (mn, 6H).

WO 97/09325 PCT/US96/1 4089 38 Examp~le 64 DIETHYL t

-AMINOCITRACONAMIDO)].

The title compound was prepared as described in Example 20, but employing a solution diethyl 2 -hydrazinopyriridine.4,5.dicarboxylate (0.20 g, 0.79 mmol) and citraconic anhydride (0.11 g, 0.95 mmol) resulting in a 77% yield (0.21 g); 'H NMR (CDCl 3 5 8.94 1H), 8.88 (bs, 6.53 111), 4.35 (in, 4H), 2.16 3H1), 1.33 (mn, 611).

Example ETHYL '-AMINO-3'-METHYLSUCCINIMIDO)]..4 To a solution of ethyl 2 -[N-(l'-aminocitraconamido)-4trifluoromethylpyrimidine5carboxylate (0.05 g, 0. 15 inmol) in EtCH (20 ml) was added Pd/C 1 The reaction was stirred under an atmosphere of H 2 overnight.

The mixture was filtered over celite and concentrated to give the title compound (0.047 g, 94% yield); 'H NMR (CDCI 3 5 9.00 I 8.39 I1H), 4.40 2H), 3. 10 (in, 2H), 2.44 (in, I1H), 1. 38 (in, 3 H).

Exampvle 66 METHYL 2- 1'-AMINo-3 '-METHYLSUCcINIMIDO)]-4- -CARB3OXYLATE The title compound was prepared as described in Example 65, but employing methyl l'-aminocitraconamido)]-4-.trifluoromethylpyimidinecarboxylate (0.02 g, 0.06 inmol), resulting in a yield of 94% (0.019 m.p. 66-68'C.

WO 97/09325 PCT/US96/14089 39 Example 67 ETHYL '-AMINOCITRACONAMIDO)]-4- 2 A solution of 5-chlorothiophene-2-carboxylic acid (16.3 g, 0.1 mole) and carbonyl diimidazole (27.8 g, 0.17 mol) in THF (200 mL) was stirred 30 minutes.

Bis(ethyl malonate)magnesium salt (14.4 g, 0.051 mol) was added and the mixture was refluxed for 7 hours, cooled to RT and concentrated. EtOAc (200 mL) and 6N HCI mL) were added and the organic layer was separated, dried (MgSO 4 and concentrated.

The ethyl 5-chloro-2-thiophenoylacetate (23 g, 0.1 mol) and N,N-dimethylformamide dimethyl acetal (12 g, 0.1 mol) were stirred for 1 hour and concentrated. To this was added, a solution of NaOEt previously prepared using sodium in EtOH [Na (2.3 g, 0.1 mol) dissolved in 500 mL EtOH] and S-methyl isothiouronium sulfate (13.9 g, 0.05 mol) that was allowed to stir for 30 minutes. The mixture was refluxed for 4 hours, cooled to RT, concentrated, dissolved in EtOAC (500 mL), washed with 4N HCI (2 x 50 mL), dried (MgSO 4 and the solvent replaced with CHC1 2 (300 mL). The solution was cooled to 0°C, mCPBA (51 g, 0.3 mol) was slowly added, and the solution was allowed to reach RT. After stirring for 2 hours, the solution was quenched by slowly pouring into a cold satd. NaHSO 3 solution (100 mL). The organic layer was then washed with satd. NaHCO 3 (100 mL), dried (MgSO 4 and concentrated. From this, the title compound was prepared as described in Example 20, but employing the ethyl 2hydrazino-4-[2'-(5'-chlorothiophene)]pyrimidie-5-carboxylate (3.0 g, 0.017 mol) {prepared according to the procedure of Example 19, but employing ethyl 2-mesyl-4- 2 '-(5'-chlorothiophene)]pyrimidine-5-carboxylate described above), resulting in a yield of 38% (1.3 m.p. 137-138°C.

WO 97/09325 WO 9709325PCTIUS96/14089 Examp~le 68 ETHYL 2-[N-(l1'-AMINOCITRACONAMIDO)-N-METHYLI..4 2 The title compound was prepared as described in Example 67 from (5'-chlorothiophene)-2-hydrazino-5-pyrimidine carboxylate (0.5 g, 1.7 mmol), but employing methyl hydrazine (0.35 g, 7.5 mmol) where hydrazine was used, resulting in a yield of 48% (0.32 GC/MS calcd for C 17 Hj 5

N

4 0 4 SC1 406, found 406.

Example 69 ETHYL 1'-AMINo-4'-METHYLPHTHALIMIDO)]-4.

The title compound was prepared as described in Example employing ethyl 2 -hydrazino-4-trifluoromethylpyrimidine-.s.carboxylate (0.20 g, 0.8 mmol) and 4-methylphthalic anhydride (0.26 g, 1.6 mmol) where citraconic anhydride was used, resulting in a yield of 29% (0.09 m.p. 50-5 1 C.

Example ETHYL I'-AMINo- 4 ',5-DICHLOROPHTHALIMIDO)]-4.

The title compound was prepared as described in Example employing ethyl 2 -hydrazino- 4 -trifluoromethylpyrimidine.s-carboxylate (0.20 g, 0.8 mmol) and 4,5-dichlorophthalic anhydride (0.3 5 g, 1.6 rnmol) where citraconic anhydride was used, resulting in a yield of 61% (0.22 m.p. 142-144'C.

WO 97/09325 PCT/US96/14089 41 Example 71 ETHYL The title compound was prepared by treating 2 carbonylchloride (Example 13, 110 mg, 0.62 mmol) with ethanol (200 mg, 4.4 mmol in 1 mL of ethyl acetate) to provide 60% of ethyl 2 -chloropyrimidine-5-carboxylate, (b) reaction of ethyl 2 -chloropyrimidine-5-carboxylate (70 mg, 0.37 mmol) with hydrazine (100 mg, 3 mmol) to afford 44% of ethyl 4 -hydrazinopyrimidine-5-carboxylate as in Example 19, reaction of ethyl 4 -hydrazinopyrimidine-5-carboxylate (0.36 g, mmol) with citraconic anhydride (0.34 g, 3.0 mmol) in analogy to Example 21 to afford 10% (0.05 g) of the title compound; m.p. 95-98 0

C

Example 72 5-BENZOYL-4-ETHYL-2-METHYLTHIOPYRIMIDINE The title compound was prepared by reaction of ethyl propionylacetate (19.4 g, 0.13 mol) and N,N-dimethylformamide dimethyl acetal (16.0 g, 0.13 mol) in analogy to Example 67, reaction with NaOEt (0.18 mol) and Smethyl isothouronium sulfate (18.7 g, 0.067 mol) also described for Example 67, (c) hydrolysis of the ethyl 4 -ethyl- 2 -methylthiopyrimidine-5-carboxylate (18.0 g, 0.08 mmol) with NaOH (12.7 g, 0.32 mol) in H20 (200 mL) as described in Example 67, (d) reaction of the 4 -ethyl- 2 -methylthiopyrimidine-5-carboxylic acid (0.50 g, 2.5 mmol) and oxalyl chloride (0.32 g, 2.5 mmol) as described in Example 11, and reaction of the 4 -ethyl-2-methylthiopyrimidine-5-carbonyl chloride (0.50 g, 2.3 mmol) and phenyl magnesium bromide as described for Example 23, resulting in a yield of 22% (0.14 g) from the 4 -ethyl-2-methylthiopyrimidine-5-carboxylic acid; 'H NMR (CDCl 3 6 9.18 1H), 7.80 2H), 7.62 1H1), 7.50 2H), 2.81 2H), 2.61 3H), 1.22 3H).

WO 97/09325 WO 9709325PCT/US96/14089 42 Example 73 ETHYL 1'-AMTNOCITRACONAMIDo)-N-METHYL] -4- The title compound was prepared as described in Example 42, but employing ethyl 1'-aminocitraconamnido)]-4-ethylpyrimidine-5-carboxylate (0.05 g, 0.16 mmol), resulting in a yield of 76% (0.04g); 'H NMR (CDCl 3 8 8.8 0 1 H), 6.46 11H), 4.30 2H1), 3.58 3H1), 3.18 (in, 2H), 2.16 311), 1.33 (in, 6H1).

Example 74 ETHYL 2-[N-ACETYL-N-( 1'-AMINOCITRACONAMIDO)] -4- Ethyl 1'-aminocitraconainido)]-4-propylpyrimidine-5-carboxylate (0.060 g, 0.19 mmole) and 1.5 mL of an acetic anhydride and pyridine (1:1 molar) solution were stirred overnight. Concentration and chromatography (SiO 2 hexanes/EtOAc, 3:2) provided the title compound (0.03 g, 44% yield); '11 NMR

(CDCI

3 6 9.0 111), 6.5 1H), 4.4 211), 3.1 2H1), 2.82 2.19 311), 1.7 (in, 2H), 1.39 311), 0.99 311).

Example ETHYL 2-[N-ACETx'L-N-( 1'-AMINOCITRACONAMIDO)]-4- The title compound was prepared as described in Example 74, but employing ethyl 2- 1'-aminocitraconamnido)] carboxylate 14 g, 0.4 mmol), resulting in a yield of 67% (0.11 'H1 NMR (CDCl 3 6 9.14 111), 6.55 111), 4.45 211), 2.83 311), 2.20 31-1), 1.39 311).

WO 97/09325 PCTIUS96/1 4089 43 Example 76 METHYL 2 -HYDRAZiNO-4-PENTAFLUOROETHYLPYRIMIDINE.

The title compound was prepared according to the procedure of Example 47 but employing a solution of methyl 2 -chloro- 4 carboxylate (0.55 g, 1.9 mmol; itself prepared by a reaction of methanol with 2-chloro- 4 -Pentafluoroethylpyrimidine-5-carbonyl chloride (see Example 17)) and hydrazine (0.3 g, 9.4 mmol), resulting in a yield of 99% (0.54 'HNMR (CDCl 3 5, 8.87 1H), 7.09 1H), 3.92 3H), 1.7 2H).

Examlie, 77 METHYL '-AMINOCITRACONAMIDO)]-4- The title compound was prepared according to the procedure of Example but employing 2 -hydrazino-4-pentafluoroethylpyrimidine.s..caboxylate (0.54 g, 1.9 mniol) and citraconic anhydride (0.25 g, 2.3 mniol), resulting in a yield of 98% (0.71 m.p. 94-95*C.

Examle 78 METHYL '-AMINOCITRACONAMIDO)]-N-METHYL].4- The title compound was prepared according to the procedure of Example 73 but employing methyl 2 -[N-(l'-aminocitraconamido)-4-pentafluoroethylpyrimidine- (0.05 g, 0.13 mmol), a base and methyl iodide (0.04 g, 0.26 mmol); 'L-NMR (CDCl 3 6 9.02 11H), 8.79 11-1), 6.49 (mn, 11H), 3.92 3H), 3.59 3H), 2.16 3H).

WO 97/09325 WO 9709325PCTIUS96/14089 44 Examnie 79 t-BUTYL 1'-AMINOCITRACONAMIDO)]-4-

TRFURMTYPRMDN--ABXLT

The title compound was prepared from t-butyl 2-hydrazino-4- (0.88 g, 3.2 mmol) (prepared as described for Example 19, but employing t-butyl 2 carboxylate) as described in Example 20, resulting in a yield of 10% 12 NMR (CDCl 3 5 8.84 111), 7. 50 1H), 6.53 1H), 2.20 314), 1.55 9H).

Examp~le METHYL 2-IiN-(1 '-AMINOCITRACONAMIDO)]-4- The title compound was prepared from methyl 2-hydrazino-4- (0.01 g, 0.42 minol) (prepared as described for Example 19, but employing methyl 2 carboxylate) as described in Example 20, resulting in a yield'of 69% (0.096 m.p.

1 18-120 0

C.

Example 81 METHYL '-AMINOCITRACONAMIDo)-N-METHYL].4-

-CARBOXYLATE

The title compound was prepared from methyl (0.20 g, 6.1 mmol) as described in Example 42, resulting in a yield of 10% (0.02 'H NMR (CDC1 3 59.05 lH), 6.49 1H), 3.91 3H), 3.62 3H), 2.18 3H).

WO 97/09325 PCT/US96/14089 Example 82 METHYL 2-HYDRAZINO-4-(2'-THIENYL)PYRIMIDINE-5-CARBOXYLATE A solution of ethyl 2 -hydroxy- 4 (2.7 g, 10.8 mmol) and NaOH (1.3 g, 32.4 mmol) in H 2 0 (25 mL) was stirred overnight then acidified to pH<2 using concentrated HC1. The precipitate was collected, dried under vacuum, and POC13 (16.5 g, 108 mmol) was added. To this was added diethylaniline (1.6 g, 10.8 mmol), and the mixture was refluxed for 30 minutes. The solution was cooled, concentrated, poured over ice and extracted with ether (200 mL).

The 2 -chloro-4-(2'-thienyl)pyrimidine-5-carboxylic acid was then dissolved in CH 2 Cl 2 mL), treated with oxalyl chloride (5.4 g, 42.7 mmol) and DMF (2 drops), and allowed to stir overnight. The solution was then concentrated, dissolved in MeOH mL) and concentrated after stirring 10 minutes. The title compound was prepared from the crude methyl 2 -chloro-4-(2'-thienyl)pyrimidine-5-carboxylate as described in Example 19, resulting in a overall yield of 6% (0.16 GC/MS calcd for CoHoN 4 0 2

S

250, found 250.

Example 83 METHYL 2-[N-(1'-AMINOCITRACONAMIDO)]-4- 2 The title compound was prepared from methyl 2-hydrazino-4-(2'- (0.16 g, 0.64 mmol) according to the procedure of Example 20, resulting in a yield of 90% (0.20 m.p. 112-113°C.

WO 97/09325 WO 9709325PCT/US96/14089 46 Example 84 ETHYL I'-AMINOCITRACONAMIDo..N.BENZYL]-4.

The title compound was prepared from ethyl 2-(hydrazino. 1'-benzyl)-4trifluoromethylpyrimidine.5.carboxylate (0.2 g, 0.78 mmol) (prepared according to the procedure of Example 19 but employing benzylhydrazine) as described in Example resulting in a yield of 12% (0.05 'H1 NMR (CDC1 3 5 9.08 1H), 7.3 (in, 5H), 6.39 1H1), 5.21 (in, 2H1), 4.37 (mn, 2H), 2.09 3H), 1.37 (in, 3H1).

Example ETHYL t -AMINOCITRACONAMIDO.*.METHYL)]-4 21 The title compound was prepared from ethyl aminocitraconamnido)]-4-(2t..thienyl)pyrimidine..5-caboxylate (0.05 g, 0.14 minol) according to the procedure of Example 42, resulting in a yield of 19% (0.01 'H NMR (CDCl 3 8 8.83 111), 7.78 (in, 11H), 7.41 (in, 11H), 7.10 (in, 1H), 6.47 111), 4.34 (in, 2H1), 3.61 3H), 2.20 3H), 1.33 (in, 3H).

Example 86 I'-AMINOCITRACONAMIDO)].4 TRIFLUOROMETHYLPYRIMIDINE..%.CARBOXYLIC

ACID

A solution of t-butyl '-aminocitraconamido)-4trifluoromethylpyrimidine5carboxylate (0.20 g, 0.5 mmol) in concentrated formic acid (8 mL) was refluxed for one hour, cooled, diluted with H 2 0 (10 mL), extracted with EtOAc (2 x 20 mL), dried (MgSO 4 and concentrated. The solid was washed with

*CH

2 C1 2 (2 x 20 mL) and dried under vacuum to give the title compound in 60% yield 10 m.p. 107-109'C.

WO 97/09325 WO 9709325PCTIUS96/1 4089 47 Example 87 t -AMINOCTTRACONAMIDO)]-4.

TRIFLUOROMETHYLPYRIMIDINE

The title compound was prepared from 5-benzoyl-2-hydrazino-4-.

trifluoromethylpyrimidine (0.15 g, 0.53 nimol) (prepared as described for Example 19, but employing 5-benzoyl- 2 -mesyl-4-trifluoromethylpyimidine) as described in Example 20, resulting in a yield of 6% (0.015 NMR (CDCI 3 8 8.5 (s,1IH), 7.8 7.65 7.5 6.5 3.45 2.2 (s,3H).

Example 88 ETHYL t -AMINOCITRACONAMIDO)].4.

3 The title compound was prepared from ethyl 2-hydrazino-4-(3- (0.22 g, 0.88 mmol) (prepared in the same manner as Example 35) as described in Example 20, resulting in a yield of 40% (0.11 m.p. 47- 48 0

C.

Example 89 ETHYL '-AMINOCITRACONAMIDO-N-METHYL)]-4 (3 The title compound was prepared from ethyl aminocitraconanfido)]-4(3'thienyl)pyrimidine-5-.carboxylate (0.05 g, 0.14 mmol) as described in Example 42, resulting in a yield of 19% (0.01 39-41'C.

WO 97/09325 WO 9709325PCT/US96/14089 48 Example ETHYL 2-[N-(l1'-AMINOCITRACONAMIDO)] -4- 2 The title compound was prepared from ethyl 2-hydrazino-4-[2'-(5'methylthienyl)]pyrimidine-5-carboxylate (0.22 g, 0.88 mmol) (prepared in the same manner as Example 67) as described for Example 20, resulting in a yield of 40% (0.11 m.p. 138-139TC.

Examp~le 91 ETHYL 1'-AMINOCITRACONAMIDO)] -4- (2'-FURANYL) The title compound was prepared by reaction of ethyl 2'flhranoylacetate (10 g, 0. 05 5 mol) with N,N-dimethylformamide dimethyl acetal (7.3 g, 0.055 mol) as described for Example 67, subsequent reaction with a solution NaOEt (0.058 mol) and S-methyl isothouronium, sulfate (7.7 g, 0.028 mol) also described for Example 67, oxidation with mCPBA (9.8 g, 0.057 mol) also described for Example 67, reaction with hydrazine (1.5 mL, 0.05 mol) as described for Example 19, and (e) reaction with citraconic anhydride (4 mL, 0.05 mol) as described for Example resulting in an overall yield of 1% (0.18 mn.p. 38-40'C.

Example 92 ETHYL 2- I'-AMINOCITRACONAMIDO-N-METHYL)].4- 2 1 The title compound was prepared from ethyl I'-methylhydrazino)-4- 2 -(5'-methylthienyl)]pyrimidine..s-carboxylate (3.0 g, 10.0 mmol) (prepared in the same manmer as Example 67 employing methyl hydrazine) as described in Example WO 97/09325 WO 9709325PCT/US96/14089 49 resulting in a yield of 40% (1.6 'H1 NMR (CDC1 3 8 8.71 7.7 6.74 6.5 4.33 3.6 (s,311), 2.5 2.2 1.32 (t,3H).

Example 93 ETHYL 2-IIN-(1 t -AMINOCITRACONAMIDO)]-4.

21 The title compound was prepared by reaction of ethyl 2'benzothienoylacetate (24.8 g, 0. 1 mol) with N,N-dimethylformamide dimethyl acetal (7.3 g, 0.055 mol) as described for Example 67, subsequent reaction with a solution of NaOEt (0.12 mol) and S-methyl isothouronium sulfate (13.9 g, 0.05 mol) also described for Example 67, oxidation with mCPBA (8.5 g, 0.05 mol) also described for Example 67, reaction with hydrazine (1.5 mL, 0.05 mol) as described for Example- 19, and reaction with citraconic anhydride (4 mL, 0.05 mol) as described for Example 20, resulting in an overall yield of 0. 1% (0.05 m.p. 165-166'C.

Example 94 METHYL 1'-AMINOCITRACONAMIDo-N-ETHYL)]-4- The title compound was prepared from methyl aminocitraconamido)]-4-trifluoromethylpyrimidine..s.carboxylate (0.20 g, 0.61 mmol) as described for Example 42, but employing ethyliodide, resulting in a yield of 18% (0.04 'H NMR (CDCl 3 6 9.01 1HI), 6.48 114), 4.05 3.87 2.14 3 1. 27 3 H).

WO 97/09325 WO 9709325PCTIUS96/I 4089 Example ETHYL I'-AMINOCITRACONAMIDO-N-BUTANOYL)]-.4.

The title compound was prepared from ethyl aminocitraconamido)I-4-trifluoromethylpyrimidine-5.caboxylate (0.10 g, 0.29 mmol) as described for Example 74, but employing butyric anhydride, resulting in a yield of 23% (0.03 'H NMR (CDCl 3 8 9.13 I 6.54 I 4.42 2H), 3.17 2H), 2.18 3H), 1.77 2H), 1.36 3H), 1.01 3H).

Example 96 ETHYL '-AMINOCITRACONAMIDO)-N-(N'-METHYLCARBOyJAM1DYL)-4.

Ethyl 1'-aminocitraconamido)]-4-trifluoromethylpyrimidine-5carboxylate (0.05 g, 0.15 mmol) and methylisocyanate (0.3 mL) were heated to for 3 minutes and allowed to cool to RT. After 30 minutes, the residual isocyanate was removed under vacuum, and the solid was washed with hexanes to provide the title compound in a 70% yield (0.03 m.p. 132-134'C.

Example 97 ETHYL 1'-AMINOCITRACONAMIDo)-N-( 1 "-oxo-2"-PHENYLETHYL]-4- A solution of -ethyl 2- rN-( I -aminocitraconamido)] -4trifluoromethylpyrimidine-5-carboxylate (0.04 g, 0.12 mmol), phenylacetylchloride (0.08 mL, 0.6 mmol) and pyridine (0.05 mL, 0.6 mmol), in CH 2 C1 2 (15 mL) was stirred hours, washed with H 2 0, dried NMgOW, concentrated and chromatographed (SiO 2 hexanes/EtOAc, 2:1) to give the title compound in 42% yield (0.021 'H NMR WO 97/09325 PCTIUS96/1 4089 51 (CDC1 3 8 9.11 1H), 7.27 (in, 511), 6.53 1H), 4.46 211), 4.42 2H), 2.17 (s, 3H), 1.37 3H).

Examvle 98 ETHYL 1'-AMINOCITRACONAMIDO)]-4-METHOXYMETHYL The title compound was prepared by reaction of ethyl methoxyketoacetate (10.5 g, 0.07 mol) with triethyl orthoformate (9.7 g, 0.07 mol) and urea (3.9 g, 0.07 mol) as described for Example 3, reaction with NaOEt (0.02 mol) as described for Example 5, reaction with POC1 3 (6.5 mL, 0.07 mol) as described for Example 7, reaction with hydrazine (2 mL, 0.07 mol) as described for Example 19, and reaction with citraconic anhydride (6 mL, 0.07 mol) as described for Example resulting in an overall yield of 8% (1.8 'H NMR (CDCl 3 6 8.87 1H), 8.2 (s, 11H), 6.5 1H), 4.87 2H1), 4.35 2H1), 3.47 311), 2.18 3H), 1.35 3H).

Example 99 ETHYL 1'-AMIN0CITRACONAMIDO)-N-(ETH0XYCARB0NYL)].4 The title compound was prepared from ethyl (0.06 g, 0.19 inmol) as described for Example 97, but employing ethyl chioroforinate, resulting in a yield of 71% (0.055 'H NMR (CDCl 3 6 9.13 6.54 lH), 4.39 (in, 4H), 2.18 (s, 3H1), 1.31 6H1).

WO 97/09325 WO 9709325PCTIUS96/1 4089 52 Examle 100 ETHYL 2-[N-(l1'-AMINOCITRACONAMIDo)-N-BENZOYL]-4.

The title compound was prepared from ethyl 1'aminocitraconamido)]-4-trifluoromethylpyrimidine-.5-carboxylate (0.09 g, 0.26 mmol) as described for Example 97, but employing benzoyl chloride, resulting in a yield of 69% (0.08 'H NMR (CDCI 3 8 8.99 lH), 7.72 2H), 7.52 (dd, IH), 7.41 (dd, 211), 6.55 111), 4.39 2H), 2.19 3H), 1.35 3H).

Example 101 1'-AMINOCITRACONAMIDO)]-4-ETHYLPYRIMIDINE The title compound was prepared by reaction of 5-benzoyl-4-ethyl-2methylthiopyrimidine 14 g, 0. 54 mmol) and mCPBA (0.28 g, 1.6 mmol) as described for Example 67, reaction with hydrazine (0.09 g, 2.7 mmol) as described for Example 19, and reaction with citraconic anhydride as described for Example resulting in a overall yield of 25% (0.046 mn.p. 49-50'C.

Example 102 2- 1'-AMINOCITRACONAMIDo)-N-METHYL]-4-ETHYLPYRIMIDINE The title compound was prepared as described for Example 42, but employing 2 -[N-(V'-aminocitraconamido)]-5-benzoyl-4-ethylpyrimidine (0.06 g, 0.18 nimol), resulting in a yield of 40% (0.025 'H NMR (CDCl 3 8 8.41 IlH), 7.7 8 (d, 2H), 7.60 (dd, lH), 7.44 (dd, 2H), 6.43 IH), 3.61 3H), 2.68 (m 2.19 31-), 1.2 (m 3H1).

WO 97/09325 WO 9709325PCT/US96/14089 53 Examnle 103 ETHYL-2-[N-(1 '-AMINOCITRACONAMIDO)-N-METHYL]-4- The title compound was prepared according to the procedure of Example 78 but employing ethyl-2-[N-( 1'-aminocitraconamide)]-4-pentafluoroethylpyrimidine..s.

carboxylate (0.1 g, 0.3 mrnol) and methyl iodide (0.07 g, 0.5 mmol) under basic conditions, resulting in a yield of 47% (0.05 'I-INMR (CDCl 3 8 9.01 1H), 8.80 (s, 11), 6.49 (in, 111), 4.39 211), 3.62 3H), 2.18 3H), 1.36 3H-).

Example 104 ETHYL 1'-AMINOCITRACONAMIDQ)]-4- 2 The title compound was prepared from ethyl 2-mesyl-4-(2'thianaphthyl)pyrimidine-5-carboxylate (2.0 g, 5.5 inmol) as described in Example 93, but employing methyl hydrazine (0.9 mL, 16.5 rmol) where hydrazine was used, this followed by reaction with citraconic anhydride (1.5 mL, 16.5 inmol) also in anology to Example 93; resulting in a yield of 69% 'H1 NMR (CDCl,) 6 8.8 IH), 8.15 IH), 7.8 (in, 7.3 (in, 2H), 6.6 111), 4.37 2H), 3.65 3H), 2.17 3H), 1.34 3H1).

Example 105 ETHYL 1'-AMINOCITRACONAMIDO)]-4- 2 -TH IAZOLYL)PYRI MI DINE- 5-CARB OXYLATE 2-Bromothiazole (8.25 g, 0.05 moles) in anhydrous ether (60 mL) is added dropwise to a solution of nBuLi (34 mnL, 1.5M solution, 0.051 rnmol) in anhydrous ether (60 mL) cooled to -78'C and stirred for 30 minutes. Carbon dioxide is bubbled into the solution and after saturation is achieved, the reaction mixture is poured WO 97/09325 PCT/US96/14089 54 over dry-ice. H 2 0 (10 mL) is added and the mixture basified to pH=9 with NaOH (1 The aqueous layer is acidified with concentrated HCI to pH <3 then extracted into ether, dried (MgSO 4 and concentrated to provide the thiazole-2-carboxylic acid in 57% yield (3.2 'H NMR (MeOD) 6 8.00 1H); 7.94 1H). The title compound was then prepared by reaction of thiazole-2-carboxylic acid (3.7 g, 29.0 mmol) and bis(ethyl malonate)magnesium salt (4.3 g, 15.0 mmol) as described in Example 67, (b) reaction of 2-thiazolylacetate (2.5 g, 12.8 mmol) and N,N-dimethylformamide dimethyl acetal (2.42 g, 12.8 mmol) as described for Example 67, reaction with NaOEt (13.8 mol) and S-methyl isothouronium sulfate (1.78 g, 6.4 mol) also described for Example 67, oxidation with mCPBA (6.1 g, 35.0 mmol) also described for Example 67, (e) reaction of ethyl 2-mesyl-4-(2'-thiazolyl)pyrimidine-5-carboxylate (1.08 g, 3.45 mmol) with hydrazine (0.33 mL, 10.3 mmol) as described for Example 19, and reaction with citraconic anhydride (0.31 mL, 3.5 mmol) described for Example 20, resulting in an overall yield of 8% (0.10 g) from ethyl 2-mesyl-4-(2'-thiazolyl)pyrimidine-5carboxylate; m.p 42-44 0

C.

Example 106 ETHYL 2-[N-(l'-AMINOCITRACONAMIDO)-N-METHYL]-4- The title compound was prepared as described in Example 20, but employing ethyl 2-(1'-methylhydrazino)-4-(2'-thiazolyl)pyrimidine-5-carboxylate (0.96 g, 3.4 mmol) (prepared according to the procedure of Example 19 but employing ethyl 2-mesyl-4-(2'-thiazolyl)pyrimidine-5-carboxylate and methyl hydrazine where hydrazine was used). resulting in a yield of 50% (0.64 'H NMR 8 8.69 (bs, 1H); 7.92 1H); 7.50 1H); 6.50 1H); 4.35 2H); 3.62 3H); 2.2 3H); 1.27 3H).

WO 97/09325 WO 9709325PCT/US96/14089 Example 107 1'-AMINOCITRACONAMIDO-N-METHYL)]-4-ETHYLPYRIMIDINE The title compound was prepared as described for Example 101, but employing 5-butanoyl-4-ethyl-2-methylthiopyrimidine (1.16 g, 5.2 mmol) (prepared as described for Example 72) where 5-benzoyl-4-ethyl-2-methylthiopyrimidine was used, resulting in an overall yield of 15% (0.24 'H NMR 8 8.80 111), 6.42 1H), 3.60 3H1), 2.65 (m 2.20 3H), 1.65 (in, 3H), 1.23 (in, 0.99 (in, 3H).

Example 108 ETHYL 2- I'-AMINOCITRACONAMIDO)]-4- The title compound was prepared as described for Example 67, but employing cyclopropanecarboxylic acid (5.5 g, 63.9 mnmol) where 5-chlorothiophene-2carboxylic acid was used, resulting in an overall yield of 0.8% (0.09 m.p. 76-78'C.

Example 109 ETHYL 2- I'-AMINOCITRACONAMIDO-N-METHYL)]-4- The title compound was prepared as described for Example 108 from cyclopropanecarboxylic acid (5.5 g, 63.9 inmol), but employing methyl hydrazine where hydrazine was used, resulting in an overall yield of 0.3% (0.062 m.p. 53- 0

C.

WO 97/09325 WO 9709325PCTIUS96/14089 56 Example 1 4 -ETHYL-5-(HYDROXYMETHYL)-2-METHYLTHIOPYRIMIDINE To a solution of ethyl 4 -ethyl- 2 -methylthiopyrimidine-5-carboxylic acid g, 10.1 mmol) (prepared as described in steps a-c of Example 72) and N-methyl morpholine (1.16 mL, 10.6 mmol) in dimethyl glycol (50 mL) at 0 0 C was added isobutyichioroformnate (1.38 mL, 10.6 mmol). The mixture was stirred 10 minutes, filtered, and cooled to 0 0 C. A solution of NaBH 4 (0.40 g, 10.6 mmol) in H 2 0 (10 ML) was added to the filtrate and the mixture was stirred for 10 minutes. H 2 0 (0 mL) Was added and the mixture was extracted with EtOAc (100 mL),washed with brine (20 mL), and concentrated to provide the title compound in 56% yield (1.04 GU/MS calcd for CgH 12 N,OS 184, found 184.

Exampnle 11 I'-AMINOCITRACONAMIDo)-N-METHYL]..4.ETHYL..$.

(HYDROXYMETHYL)PYRIMID1NE A solution of 5-hydroxymethyl-4-ethy1-2-methylthiopyrimidine (0.50 g, 2.72 mmol), t-butyldimethylsilylchloride (0.49 g, 3.26 mmol), and imidazole (0.20 g, 2.99 mmol) in DMF (5 mL) was stirred overnight and concentrated to give of butyldimethylsiloxymethyl-4.ethyl-2methylthiopyrimidine in a 94% yield (0.76 g); GC/MS calcd for C 14

H

2 6

N

2 OSSi 299, found 299. The title compound was then prepared by oxidation of 5-t-butyldimethylsiloxymethyl-4-ethyl.2 methylthiopyrimidine (0.30 g, 1.0 mmol) with mCPBA (0.35 g, 2.0 mmol) as described for Example 67, reaction with methyl hydrazine (0.16 mL, 3.0 mmol) followed by citraconic anhydride 11 g, 1 .0 mmol) also described in Example 67; reaction of 2l'-aminocitraconamido)-5t-butyldimethylsiloxymethyl-4ethylpyrimidine (0.08 g, 0.21 mmol) and concentrated HCI (0.5 mL) in EtOH (20 mL) for 1 hour, concentration and chromatography (Hexanes/EtOAc, 2: 1) to provide the title compound in 76% yield (0.043 g) from 2- I -aminocitraconamido)]-5-t-butyldimethylsiloxymethyl-4 ethylpyrimidine;m.p. 108-10OOC.

WO 97/09325 WO 9709325PCT/US96/14089 57 Example 112

(METHOXYMETHYL)PYRIMIDINE

A solution of 4 -ethyl-5-hydroxymethyl-2-methylthiopyrimidine (0.63 g, 3.42 mmol), Ag 2 0 (1.6 g, 6.9 mmol) and Mel (1.1 mL, 17.1 mmol) in CH 3 CN (10 mL) was stirred overnight, filtered and concentrated to give 4 -ethyl-5-methoxymethyl-2methyithiopyrimidine in 98% yield (0.66 GC/MS calcd. for C 9

H,

4

N

2 0S 198, found 198. The title compound was then prepared by oxidation of methoxymethyl-2-methylthiopyrimidine (0.66 g, 3.35 mmol) with mCPBA (1.18 g, 6.85 mmol) as described for Example 67, and reaction with methyl hydrazine (0.36 mL, 6.85 mmol) followed by citraconic anhydride (0.77 g, 6.85 mmol) also described in Example 67; resulting in a yield of 41% (0.40 g) from 4-ethyl-5-methoxymethyl-2methylthiopyrimidine; 'H NMR (CDCl3) 8 8.14 1 6.43 I1H), 4.29 2H), 3.53 3H1), 3.36 3H), 2.68 (in, 2H1), 2.14 3H), 1. 13 (in, 3H).

Example 113 1 -AMINOCITRACONAMIDO)]-5-(METHOXYMETHYL)-4.

TRIFLUOROMETHYLPYRIMIDINE

The title compound was prepared as described for Example 112, but employing of 4 -trifluoromethyl-5-hydroxymethyl-2-methylthiopyrimidine (0.50 g, 2.23 minol) (prepared in analogy to Example 110) where 4-ethyl-5-hydroxymethyl-2methyithiopyrimidine was used, and employing hydrazine (0.09 mL, 2.87 rnmol) where methyl hydrazine was used, resulting in an overall yield of 35% (0.25 'H NMR (CDC13) 8 8.63 1H), 8.20 1H), 6.48 11H), 4.46 2H), 3.75 3H), 2.12 (s, 3H).

WO 97/09325 WO 9709325PCTIUS96/14089 58 Example 114 l'-AMINOCITRACONAMIDO)]-5-(ETHOXYMETHYLCARBONATE)-4.

TRIFLUOR0METHYLPYRIMIDINE To a solution of 2 -IIN-('-amninocitraconamido)]-4acid (0.10 g, 0.317 mmol) and Nmethylmorpholine (0.03 5 mL, 0.317 rm-ol) in THF (10 mL) at 0 0 C was added ethyichioroformate (0.030 mL, 0.317 mmol). After stirring 5 minutes, NaBH 4 (0.040g, 1.05 mmol) was added, MeOH (7 mL) was added, and the mixture was stirred minutes then concentrated. The oil was dissolved in EtOAc (30 mL) and washed with I N HCl (10 mL), 1 N NaHCO 3 (10 mL), and brine (10 mL), dried (MgSO 4 concentrated and chromatographed (Si0 2 hexanes/EtOAc, 2:1) to provide the title compound in 19% yield (0.022 'H NMR (CDC1 3 8 9.03 1H), 6.53 11H), 4.88 4.36 2H), 2.19 3H), 1.32 3H).

Example 115 ETHYL 1'-AMINOCITRACONAMIDO)]-2-PHENYLPYRIMIDINE-.5..CARBOXYLATE The title compound was prepared by reaction of diethylethoxymethylenemnalonate (31 g, 144 rmol) with benzamidine (22 g, 144 mmol) under basic conditions to afford ethyl 2 -phenyl-4-hydroxypyrimidine-5-carboxylate in 64% yield in analogy to Example 15, reaction of ethyl 2-phenyl-4-hydroxypyrimidine-5carboxylate (1.5 g, 6 mmol) and POC1 3 (9.4 g, 62 rnmol) to afford 8 1% of ethyl 2in analogy to Example 7, reaction of ethyl 2 -phenyl-4-chloropyrimidine-5-carboxylate (12 g, 46 mmol) with hydrazine (4.4 g, 137 mimol) to afford 99% of ethyl 4 -hydrazino-2-phenylpyrimidine-5-carboxylate in analogy to Example 18, reaction of ethyl 4-hydrazino-2-phenylpyrimidine-5carboxylate (12 g, 46 mmol) with citraconic anhydride (10 g, 92 mmol) to afford (12 g) of the title compound 155-156*C) in analogy to Example 21.

WO 97/09325 WO 9709325PCTIUS96/14089 59 Example 116 ETHYL-4-[N-( 1'-AMINQCITRACONAMIDO)-N-METHYL...% The title compound was prepared according to the procedure of Example 68 but employing ethyl I'-aninocitraconamide)I-4-phenylpyrimidine-..

carboxylate (0.1 g, 0.3 mmol) and methyl iodide (0.09 g, 0.7 mmol) under basic conditions, resulting in a yield of 45% (0.05 m.p. 1 18-1 19'C.

Example 117 ETHYL 4-[N-ACETYL-N-( 1'-AMINOCITRACONAMIDo)]-2- The title compound was prepared according to the procedure of Example but employing ethyl I'-aminocitraconamide)]-4-phenylpyrimidine..s carboxylate (0.5 g, 1.3 mmol, prepared in Example 115) and acetic anhydride (5 mL) under basic conditions, resulting in a yield of 10% (0.05 'HNMR (CDCl 3 5 9.15 (d, I1H), 8.19 211), 7.47 (in, 3H1), 6.70 I1H), 4.42 2H), 2.28 6H1), 1.43 3H).

Examiple 118 ETHYL 1'-AMINOCITRACONAMIDO)]-2- The title compound was prepared by reaction of diethylethoxymethylenemnalonate (6 g, 28 mmol) with acetamidine hydrochloride (3 g, 31 mnmol) to afford 67% of ethyl 2 -methyl-4-hydroxypyrimidine-5-carboxylate in analogy to Example 15, reaction of ethyl 2-methyl-4-hydroxypyrimidine-5carboxylate (2.8 g, 15 mmol) with POCl 3 (46 g, 300 mmol) to afford 28% of ethyl 4- WO 97/09325 WO 9709325PCT/US96/14089 reaction of ethyl 4-chloro-2- (0.25 g, 1.25 rnmol) with hydrazine (0.2 g, 6.3 mmol) to afford 96% of 4 -hydrazino-2-methylpyrimidine-5-carboxylate in analogy to Example 18, reaction of 4 -hydrazino-2-methylpyimidine-..car.boxylate (0.24 g, 1.2 mmol) with citraconic anhydride (0.16 g, 1.44 mmol) to afford 70% (0.24 g) of the title compound 98-99*C) in analogy to Example 21.

Example 119 ETHYL 1'-AMINOCITPACONAMIDO)]-2-.

The title compound was prepared by reaction of diethyl ethoxymethylenemnalonate (11I g, 52 mmol) with phenylacetamidine (8 g, 60 rnmol) to afford 40% of ethyl 2 -benzyl-4-hydroxypyrimidine-5-carboxylate in analogy to Example 15, reaction of 2 -benzyI- 4 -hydroxypyrimidine-5-carboxylate (3 g, 12 mmol) with POCl 3 (18 g, 116 mmol) to afford 45% of 2 carboxylate in analogy to Example 7, reaction of 2 carboxylate (1.5 g, 5.5 mmol) with hydrazine (0.5 g, 16 mnmol) to afford 75% of 2in analogy to Example 18, reaction of ethyl 2 -benzyl-4-hydrazinopyrimidine5carboxylate (1g, 3.5 mmol) with citraconic anhydride (1g, 11I minol), in analogy to Example 2 1, to afford 70% (0.9 g) of the title compound; m.p. 117-1 18*C.

Example 120 ETHYL I'-AMINOCITRACONAMIDO)]-2- (3 The title compound was prepared by reaction of diethyl ethoxymethylenemnalonate 5 g, 24 mmol) with 3-nitrobenzamidine (5 g, 25 mmnol) to afford 86% of ethyl 4 -hydroxy- 2 -(3'-nitrophenyl)pyrimidine-5-carboxylate in analogy WO 97/09325 PCT/US96/14089 61 to Example 15, reaction of ethyl 4 -hydroxy-2-(3'-nitrophenylpyimidine-5.

carboxylate (6 g, 21 mmol) with (chloromethylene)dimethylammonium chloride (4 g, 31 mmol) concentration of reaction mixture, followed by hexane and ether wash afford of ethyl 4-chloro-2-(3'-nitrophenyl)pyrimidine-5-carboxylate, reaction of ethyl 4 -chloro- 2 -(3'-nitrophenyl)pyrimidine-5-carboxylate (1.9 g, 6 mmol) with hydrazine (0.6 g, 18 mmol) to afford 33% of ethyl 4 -hydrazino-2-(3'-nitrophenyl)pyrimidine-5carboxylate in analogy to Example 18, reaction of ethyl 4-hydrazino-2-(3'- (1.8 g, 6 mmol) with citraconic anhydride in analogy to Example 21, to afford 80% (0.5 g) of the title compound; m.p. 1 18-120*C.

Example 121 ETHYL '-AMINOCITRACONAMIDO)]-2- 41 The title compound was prepared by of diethyl ethoxymethylenemalonate (6.3 g, 29 mmol) with 4 -trifluoromethylphenylbenzamidine g, 29 mmol) to afford 50% of ethyl 4-hydroxy-2-(4'in analogy to Example 15, reaction of ethyl 4 -hydroxy-2-(4'-trifluoromethylphenyl)pyrimidine-5-caboxylate (3 g, 9.6 mmol) with POCd 3 (49 g, 322 mnol) to afford 97% of ethyl 4-chloro-2-(4'in analogy to Example 7, reaction of ethyl 4 -chloro- 2 4 '-trifluoromethylphenyl)pyrimidine-5-carboxylate (2 g, 6 mmol) with hydrazine (0.6 g, 18 mmol) to afford 60% of ethyl 4-hydrazino-2-(4'trifluoromethylphenyl)pyrimidine 5-carboxylate in analogy to Example 18, reaction of ethyl 4 -hydrazino- 2 4 '-trifluoromethylphenyl)pyrimidine-5-carboxylate (1 g, 3 mmol) with citraconic anhydride (I g, 9 mmol) to afford 40% (0.5 g) of the title compound 148-150'C) in analogy to Example 21.

WO 97/09325 WO 9709325PCT/US96/14089 62 ExaMple 122 ETHYL 1'-AMINOCITRACONAMIDO)]-2..

2t The title compound was prepared by reaction of diethyl ethoxymnethylenemnalonate (8 g, 35 mmol) with thienyl-2-formamnidine hydrochloride (5.7 g, 35 minol) to afford 74% of ethyl 4 -hydroxy-2-(2'-thienyl)pyrimidine-5carboxylate in analogy to Example 15, reaction of ethyl 4-hydroxy-2-(2'- (2 g, 8 mmol) with POC1 3 (24 g, 160 mmol) to afford 96% of ethyl 4 -chloro- 2 -(2'-thienyl)pyrimidine-5-carboxylate in analogy to Example 7, reaction of ethyl 4-chloro-2-(2 t -thienyl pyrimidine-5-carboxylate (0.57 g, 2.2 mmol) with hydrazine (0.34 g, 11I mmol) to afford 99% of ethyl 4-hydrazino-2-(2'-thienyl)in analogy to Example 18, reaction of ethyl 4-hydrazino- 2 2 t -thienyl)pyrimidine-5-carboxylate (0.55 g, 2.1 mmol) with citraconic anhydride (0.71 g, 6.3 mimol) in analogy to Example 21 to afford 80% (0.61 g) of the title compound; m.p. 164-164 0

C.

Example 123 ETHYL t -AMINOCITRACONAMIDO)-N-METHYL]..2 21 The title compound was prepared by reaction of ethyl 4-chloro-2-(2'- (0.3 g, 1.1 mmol)) with methylhydrazine (0.25 g, 5.6 mmol)) to afford 99% of ethyl 4 -(l'-methylhydrazino)-2-(2'-thienyl)pyrimidine-5carboxylate in analogy to Example 18, reaction of ethyl I'-methylhydrazino)-2- 2 -thienyl)pyrimidine-5-carboxylate (0.3 g, 1 nunol)) with citraconic anhydride (0.4 g, 3 mmol) in analogy to Example 21 to afford 52% (0.21 g) of the title compound; m.p.

126-127 0

C.

WO 97/09325 PCT/US96/14089 63 Example 124 ETHYL '-AMINOCITRACONAMIDO)]-2-(3',4'-DICHLOROBENZYL)- The title compound was prepared by of diethyl ethoxymethylenemalonate (6 g, 27 mmol) with dichlorophenylacetamidine (5.5 g, 27 mmol) to afford 37% of ethyl 4-hydroxy-2-(3',4'-dichlorobenzyl)pyrimidine-5carboxylate in analogy to Example 15, reaction of ethyl 4-hydroxy-2-(3',4'- (2 g, 6 mmol) with POCI 3 (14 g, 92 mmol) to afford 62% of ethyl 4-chloro-2-(3',4'-dichlorobenzyl)pyrimidine-5-carboxylate in analogy to Example 7, reaction of ethyl 4 -chloro- 2 3 ',4'-dichlorobenzyl)pyrimidine- (1 g, 2.8 mmol) with hydrazine (0.27 g, 8.3 mmol) to afford 99% of ethyl 4-hydrazino-2-(3',4'-dichlorophenyl)pyrimidine-5-carboxylate in analogy to Example 18, reaction of ethyl 4-hydrazino-2-(3',4'-dichlorobenzyl)pyrimidine-5-carboxylate (0.9 g, 2.7 mmol) with citraconic anhydride (0.49 g, 4.4 mmol) in analogy to Example 21 to afford 47% (0.3 g) of the title compound; m.p. 119-121 C.

Example 125 SYNTHESIS OF REPRESENTATIVE

COMPOUNDS

BY COMBINATORIAL CHEMISTRY

TECHNIQUES

This example illustrates the synthesis of a representative class of compounds of this invention, 2 -substituted-4-trifluoromethyl-5-pyrimidine carboxylic acid methyl esters, by combinatorial chemistry. It should be understood that, while a specific class of compounds are illustrated in this example, the following procedure may be employed to synthesize other compounds of this invention.

A mixture of TentaGel resin (TentaGel S PHB, Advanced Chem Tec Louisville, Kentucky, 17 g, 0.2 mmol/g reactive sites) containing a free hydroxy as the reactive site and DMF (75 mL) was stored for 0.25 h. Then a solution of 2-chloro-4trifluoromethyl pyrimidine-5-carbonyl chloride (2.20 g, 10.2 mmol) in DMF (15 mL) was added. The mixture was gently shaken for 3 h and filtered. The resin was washed WO 97/09325 PCT/US96/14089 64 thoroughly with DMF (3 X100 mL) and CH 2 C1 2 (3 X100 mL) and then dried under vacuum. The resin was divided into 80 equal portions and placed into 80 separate reaction vessels (dispersion tubes). The dispersion tubes were placed into separate test tubes each containing a different amine (2.5 mmol, Appendix I) and 2 mL of a 0.1M solution of pyridine/DMF (5 molar equivalents of each amine in each test tube). The entire set of 80 reaction vessels was gently shaken for 5 hours to ensure complete reaction. Then each of the dispersion tubes was removed from the test tube containing the amine and rinsed separately to remove any unreacted materials. The dispersion tubes were then dried and submersed into 80 new test tubes (previously tared), each containing a solution of NaOMe (2.5 mL, 0.012 M solution, 0.03 mmol). The reaction was allowed to proceed overnight at room temperature under N 2 Then the dispersion tubes were removed from the solution and individually rinsed with MeOH. The individual MeOH solutions were concentrated in the tared test tubes to provide known amounts of the desired 80 individual methyl esters substituted with different groups at the 2-position. Each compound was >85% pure by HPLC and had the correct molecular weight as determined by GC/MS.

Example 126 INHIBITION OF THE ACTIVATION OF NFKB AND AP-1 A. NFKB ASSAY Stable human Jurkat T-cells containing an NFKB binding site (from the MHC promoter) fused to a minimal SV-40 promoter driving luciferase expression were used in this experiment. Cells were split to 3 x 105 cells/mL every 2-3 days (cell concentration should not exceed 1 x 106 cells/mL to keep the cells proliferating in log phase). These cells were counted, resuspended in fresh medium containing Serum-Plus at a density of 1 x 106 cells/mL and plated in 96 well round bottom plates (200 iA per well) 18 hours prior to starting the experiment.

Compounds of this invention, dissolved in dimethyl sulfoxide 0.33 and 0.03 utg/mL), were then added to the 96 well plates containing the cells and the WO 97/09325 PCT/US96/14089 plates were incubated for 0.5 h at 37 0 C. Then 50 ng/mL of phorbol 1 2 -myristate-13acetate (PMA) and 1 .g/mL of phytohemagglutinin (PHA) were added to each well and the cells were incubated for an additional 5 h at 37 0 C. The plates were centrifuged at 2200 RPM for 3 minutes at room temperature and then the medium was removed. To each well was added 60 4L of cell lysis buffer and the plates were left at room temperature for 0.25 h. Then 40 pL of each cell extract was transferred to a black 96 well plate and 50 uL of luciferase substrate buffer was added. Luminescence was immediately measured using a Packard TopCount.

B. AP-1 ASSAY For AP-1, the assay was run as described above for NFB except stable Jurkat T-cells were used that contained a collagenase promoter driving luciferase expression. In addition, the concentration of PMA used was 5 ng/mL.

C. RESULTS The results of the above assays for a representative compound of this invention, ethyl 2-[N-(l'-aminocitraconamido)]-4-pentafluoroethylpyrimidine-5carboxylate (see Example 48), as percent inhibition versus control are presented in Figure 3. This figure also indicates activity of p-actin which was employed in these assays as a control cell line indicating effects on transcription. The lack of p-actin activity evidences selectivity of the test compounds for the transcription factors AP-1 and NFKB.

Expressed as ICs,'s, the results of these assays on additional test compounds are summarized in Table 3 below. The IC 50 values reported in Table 3 are the average of the measurements obtained from the NFKB and AP-1 assays described above.

WO 97/09325 PCT/US96/14089 66 Table 3 Ability of Representative Compounds of Structure to Inhibit NFKB and AP- Test IC 5 0 Test IC 50 Test IC 5 0 Compound (PM) Compound (tM) Compound

(PM)

(Example No.) (Example No.) (Example No.) 0.7 79 0.2 103 0.4 21 0.5 81 0.1 104 0.2 23 5-10 83 0.08 105 2.4 36 0.15 84 0.45 106 1.7 37 0.15 85 0.02 107 38 0.04 87 0.55 108 1.0 88 0.5 109 0.2 41 3.9 89 0.2 111 4.9 42 5 90 0.06 112 0.6 44 10-30 91 1.1 113 46 4.0 92 0.3 114 1.1 48 0.09 93 0.8 115 0.03 1.0 94 1.1 116 1.4 52 2.0 95 0.3 117 4 64 1.0 96 0.7 118 17 68 0.4 97 0.9 119 7 73 0.05 98 6.0 120 8 74 0.45 99 0.4 121 0.4 0.4 100 0.8 122 0.02 77 1.3 101 0.6 123 0.8 78 0.15 102 0.4 124 4 Based on the above results, representative compounds of this invention were found to be effective at inhibiting the activation of transcription factors NFKB and AP-1) involved in gene transcription, and therefore have utility as, for example, immunosuppressive agents.

Example 127 INHIBITION OF CYTOKINES To determine the effects of compounds on PMA/PHA-induced cytokine production, supematants from either the NFKB (for IL-8) and AP-1 (for IL-2) reporter WO 97/09325 PCTfUS96/14089 67 gene assays of Example 55 were collected and saved. Cytokine levels in the supernatants (25-50 gL aliquots) were determined by ELISA. The results of this experiment for a representative compound of this invention, ethyl (see Example 48), is presented in Figure 4 (expressed as percent inhibition versus control).

Example 128 ACTIVITY OF REPRESENTATIVE COMPOUND IN GRAFT VS. HOST AND CONTACT SENSITIVITY MODELS The murine popliteal lymph node (PLN) assay is a graft vs. host model that predicts activity of compounds in blocking human transplant rejection. The delayed-type hypersensitivity response to oxazolone is a standard contact sensitivity model. Both of these models are used routinely to evaluate compounds that are used clinically. For example, cyclosporin and cyclophosphamide are active in these models and are used clinically (Morris et al., Transplantation Proceedings 2 2(Suppl. 1):110- 112, 1990).

A. POPLITEAL LYMPH NODE MODEL Spleens are removed from donor BALB/c mice and splenocytes are isolated then irradiated (3,000 rads) to prevent donor cell proliferation. After washing and adjusting cell density, 2.5x10 6 cells are injected subcutaneously into the left hind footpad of C3H mice. On day 4, the mice are sacrificed and left popliteal lymph nodes (PLNs) weighed.

A representative compound of this invention is administered once daily by intraperitoneal injection beginning one day before footpad injection (day 0) through day 4. The compound is suspended, immediately prior to use, at a concentration of mg/mL in 0.25% methyl cellulose (Sigma) using a glass-Teflon homogenizer. For doses of 10, 20 and 30 mg/kg, appropriate dilutions of the stock solution are made so that 0.1 mL/10 g body weight is administered by intraperitoneal injection.

68 B. DELAYED TYPE HYPERSENSITIVITY

STUDY

On day 0, oxazolone (100 mL of a 3% solution) is applied to the shaved abdomen of mice. On day 7, a challenge application of oxazolone is applied (10 mL) around the right ear. A representative compound of this invention is administered from days -2 to 7 by intraperitoneal injection. The injectable solution is prepared immediately prior to use by suspending the compound in 0.25% methyl cellulose (Sigma) using a glass-Teflon homogenizer. For each dose, 0.1 mL/10 g body weight of the suspension is administered. The compound is prepared at the highest concentration for this study and appropriate dilutions of the stock solution are made so that 0.1 g body weight is administered. Twenty four hours later, the difference in right vs. left ear thickness is measured.

see* It will be appreciated that, although specific embodiments of this invention have been described herein for purpose of illustration, various modifications 15 may be made without departing from the spirit and scope of the invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group *of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims (28)

1. A compound having the structure: R N N including pharmaceutically and prophylactically acceptable salts thereof, wherein R, is R 2 a, when R 4 is R 4 and R, is R'b when R 4 is R 4 b; 9 Rb and R 4 are selected from hydrogen, halogen and an unsubstituted or substituted C 1 8 alkyI, C6- 2 aryl, C 7 12 aralkyl, C 31 2 heterocycle or C 4 16 heterocyclealkyl; R 2 and R 4 b are selected from the following chemical moieties: R9 0 A.0 I Rio R9 0 R9 0 N N I i i 9o aH0 Rio R, is selected from -CH 2 O{C(=O)O} 0 1 R 7 -C(=O)0R 7 and Ris selected from hydrogen, -CH 3 -CH 2 C 6 H 5 -F and -CF 3 R 7 is selected from hydrogen and a unsubstituted or substituted C 1 8 alkyl, Cl 1 2 aryl and C7, 2 aralkyl; R, is an unsubstituted or substituted 8 alkyl, C 61 2 aryl or C 7 12 aralkyl; R 9 is selected from hydrogen and an unsubstituted CI 8 alkyl or C 7 4 aralkyl, wherein D is a direct bond, or -NH-; RIO and are the same or different and independently selected from hydrogen and an unsubstituted or substituted C, 1 8 alkyl or C1 12 aryl; and n is an integer from 0 to 4 and each occurrence of A is a substituent 1!,ependently selected from halogen, -OH, -OR, -COOH, -COOR, -COR, -CONH 2 WO 97/09325 PCT/US96/14089 -NH 2 -NHR, -NRR, NO 2 -SH, -SR, -SOOR, -SO 3 R and -SOR, where each occurrence of R is independently selected from an unsubstituted or substituted C. 8 alkyl, C 6 2 aryl or C 712 aralkyl.

2. The compound of claim 1 wherein R 2 is R 2 a, R 4 is R 4 a, and R 5 is -C(=O)OR 7

3. The compound of claim 2 wherein R 2 a is selected from one of the following structures: R9

4. following structures: The compound of claim 2 wherein R 2 a is selected from one of the CH 3 O 0 H N C,H, N 0 N 0 -(CH 2 )1-2CH 3 -C 2 F 3 The compound of claim 2 wherein R4, is selected from -Cl, -CH 3 x S and wherein X, Y and Z are the same or different, and independently selected from hydrogen, -OH, -OR, -COOH, -COOR, -COR -CONH 2 -NH 2 -NHR, -NRR, -NO 2 -SH, WO 97/09325 PCT/US96/14089 71 -SR, -SOOR, -SO 3 R and -SOR, where each occurrence of R is independently selected from an unsubstituted or substituted C 1 .alkyl, C 6 1 2 aryl, C 7 1 2 aralkyl, C3. 12 heterocycle or C 4 -16heterocyclealkyl.

6. The compound of claim 2 wherein R is selected from hydrogen, -CF 3 and -CH 3

7. The compound of claim 2 wherein R 7 is selected from hydrogen, -CH 3 and -CH 2 CH 3

8. The compound of claim 2 wherein R 9 is selected from hydrogen, -CH -CH 2 CH 3 and -CH 2 C 6 H,.

9. The compound of claim 2 wherein RIo and are independently selected from hydrogen, -CH 3 -CF 3 -(CH 2 ),.sCH 3 -C 6 H, and -CH 2 C 6 H The compound of claim 1 wherein R 2 is R2b, R 4 is R 4 b, and R 5 is -C(=O)OR 7

11. following structures: The compound of claim 10 wherein R 4 b is selected from one of the R9 N and

12. following structures: The compound of claim 10 wherein R 4 b is selected from one of the WO 97/09325 PCT/US96/14089 72 -CH 3 0 C(CHH)1-2CH, C3 wherein X, Y and Z are the same or different, and independently selected from hydrogen, -OH, -OR, -COOH, -COOR, -COR, -CONH,, -NHR, -NRR, -NO2, -SH, -SR, -SOOR, -SOR and -SOR, where each occurrence of R is independently selected from an unsubstituted or substituted Cz.,alkyl, C6-128ff a 7-1aralkyl, C3-12heterocycle or C 1 0heterocyclealkyl.

14. The compound of claim 10 wherein Rs is selected from hydrogen, -CF, and -CHH The compound of claim 10 wherein R, is selected from hydrogen, -CH3 and -CHCCHH 3 00

16. The compound of claim 10 wherein R, is selected from -Cl, -CFhydrogen, -CH, -CH -(CH 2 1 and -CH, C 2 FH, and 4 wher17. The compouX, Y and ofZ are the same or diffwerein Rt, and are independently selected from hyselected from hydrogen, -COH, -COR, -CORCH, -CONH 2 -NH 2 d -NHR, -NRR, N 2 -SH,.H, -SR, -SOOR, -SO 3 R and -SOR, where each occurrence of R is independently selected from an unsubstituted or substituted C 18 alkyl, C 6 2 aryl, C 7 12 aralkyl, C 3 4 2 heterocycle or C 4 16 heterocyclealkyl. 14. The compound of claim 10 wherein R 6 is selected from hydrogen, -CF 3 and -CH 3 15. The compound of claim 10 wherein R 7 is selected from hydrogen, -CH, and -CH 2 CH 3 16. The compound of claim 10 wherein R(9 is selected from hydrogen, -CH 3 -CH,CH 3 and -CH 2 C 6 H 5

17. The compound of claim 10 wherein Rio and are independently selected from hydrogen, -CH 3 -CF 3 -(CH 2 1 5 CH 3 -C 6 H, and -CH 2 C 6 H 5

18. The compound of claim I wherein the compound is selected from ethyl I'-aminocitraconamido))-4-trifluoromethypyriidine.5caboxylate; ethyl 1'- 5-acetyl-2-(N-( 1'- ethyl I'-ainino-3'- ethyl 1'-amino-3 ethyl 1- ethyl 1'- amino-3'-phenylmaleimido))-2-trifluoromethylpyrimidine-5-carboxylate; ethyl 1'- amino-3', 4'-dimethylmaleimido))-2-trifluoromethylpyrimidine-5-carboxylate; ethyl 1'- ethyl 1'- ethyl 1'- 9 aminocitraconamido))-4-phenylpyrimidine-5-carboxylate; methyl I' aminocitraconamido))-4-(3'-pyridyl)pyriniidine-5-carboxylate; diethyl 1'- 9 ainiinocitraconaniido))pyrimidine-4,5-dicarboxylate; ethyl I'-aminocitraconamido))-4- 2 -thienyl)pyrimidine-5-carboxylate; ethyl I'-aminocitraconaniido)-N-methyl)-4- methyl I'-axinocitraconamido))-4-(2'- thienyl)pyrimidine-5-carboxylate; ethyl I'-aminocitraconainido)-N-methyl)-4-(2'- ethyl 1'-aminocitraconamido))-4-(5'-methyl-2'- ethyl I'-aminocitraconamido))-2-phenylpyrimidine- and ethyl 4-(N-(l1'-aininocitraconamido))-2-(2'-thienyl)pyrimidine-5- carboxylate.

19. A composition comprising a compound of any one of claims 1-18 and a pharmaceutically or prophylactically acceptable carrier or diluent. Use of a compound of any one of claims 1- 18 for the manufacture of a medicament for treating an inflammatory condition. P:\OPER\PDB\70130-96 specidoc-A)9/(X) -74-

21. The use of claim 20 wherein the inflammatory condition is an immunoinflammatory condition.

22. The use of claim 21 wherein the immunoinflammatory condition is selected from rheumatoid arthritis, osteoarthritis, transplant rejection, sepsis, ARDS and asthma.

23. The use of claim 21 wherein the immunoinflammatory condition is rheumatoid arthritis.

24. The use of claim 20 wherein the inflammatory condition is an autoimmune disease. 10 25. The use of claim 24 wherein the autoimmune disease is selected from multiple sclerosis, psoriasis, inflammatory bowel disease, glomerulonephritis, lupus, uveitis and chronic hepatitis.

26. The use of claim 20 wherein the inflammatory condition is selected from trauma, oxidative stress, cell death, irradiation damage, ischemia, reperfusion, cancer and viral infection.

27. The use of claim 20 wherein the inflammatory condition is transplant rejection.

28. A method for preventing and/or treating an inflammatory condition in a mammal in need thereof comprising administering to said mammal an effective amount of a compound according to any one of claims 1-18.

29. The method of claim 28 wherein the inflammatory condition is an immunoinflammatory condition. The method of claim 28 wherein the immunoinflammatory condition is selected from rheumatoid arthritis, osteoarthritis, transplant rejection, sepsis, A RDS and asthma. 4- S-00;15: 32 13/ 19 F:DOPERWD'O 130-6 i pcaddsnc.iAnlKU

31. The method of claim 28 wherein the imrnunoinflammatory condition is rheumatoid arthritis.

32. autoimmune disease. The method of claim 28 wherein the inflammatory condition is an

33. The method of claim 32 wherein the autoimmune disease is selected from multiple sclerosis, psoriasis, inflammatory bowel disease, glomerulonephritis, lupus, uveitis and chronic hepatitis.

34. The method of claim 28 wherein the inflammatory condition is selected from trauma, oxidative stress, cell death, irradiation damage, ischemia, reperfusion, cancer and viral infection. 9 *9 99 9 9 9 9 9 999*99 9999 9

999. 999 9 9.9. 9 9999 35. transplant rejection. The method of claim 28 wherein the inflammatory condition is 36. A compound according to claim 1, substantially as hereinbefore described. 37. A composition according to claim 19, substantially as hereinbefore described. 38. A use according to claim 20, substantially as hereinbefore described. 39. A method according to claim 28, substantially as hereinbefore described. DATED this 29th day of August, 2000 Signal Pharmaceuticals, Inc. By DAVIES COLLISON CAVE Patent Attorneys for the Applicants 04/09 '00 MON 15:23 [TX/RX NO 5717] o050